Use of GSTP1

ABSTRACT

The invention discloses the use of glutathione S-transferase Pb (GSTP1) for the prevention or treatment of cardiomyopathies or ischemic heart diseases and for the diagnosis thereof.

This is a continuation application of U.S. patent application Ser. No.13/505,078, filed on Oct. 28, 2010 which is the U.S. national stage ofInternational application PCT/AT2010/000408, filed Oct. 28, 2010designating the United States and claiming priority to Europeanapplication no. EP 09174692.5, filed Oct. 30, 2009.

The present invention relates to the use of Glutathione-S-transferaseP1.

The glutathione S-transferases (GSTs) are a multigene family of isozymesthat catalyze the nucleophilic attack of the sulfur atom of glutathione(GSH) on electrophilic groups of substrate molecules. GSTs are known asdetoxicating enzymes which conjugate toxic substances with GSH to becomemore water-soluble products which can be metabolised in the liver andfinally excreted out of the body. On the basis of the amino acidsequence, the mammalian GSTs are divided into six classes: alpha, mu,omega, pi, theta and zeta. Among these isozymes, glutathioneS-transferase P1 (GSTP1; GST-pi, GSTP1-1) is the most prevalent one inmammalian cells. GSTP1 has also generated interest as a neoplasticmarker because it is overexpressed in a variety of different humanmalignancies, including lung, colon, stomach, esophagus, mouth, kidney,ovary and testicular cancers. Most prostate cancer types containdecreased levels of GSTP1 polypeptide relative to normal prostate tissuedue to hypermethylation of the GSTP1 promoter resulting in decreasedtranscription of GSTP1 (US 2009/0186360 A1). GSTP1 is determinant incellular response to oxidative stress and protects tumor cells fromapoptosis elicited by a variety of cytotoxic agents, such as H₂O₂, UV,cisplatin, adriamycin, etoposide, thiotepa, chlorambucil, ethacrynicacid and arsenic trioxide. GSTP1 is therefore considered as a promisingtarget in tumour medicine, both as a target for drug action and as atumor marker.

GSTP1 also participates in the regulation of stress signalling andprotects cells against apoptosis by the mechanisms related to itsnon-catalytic and ligand-binding activities. For example, GSTP1 preventsLPS-induced excessive production of pro-inflammatory factors and playsan anti-inflammatory role in response to LPS. GSTP1 expression, both attranscriptional and translational levels, is up-regulated by LPSstimulation. GSTP1 also functions as an endogenous inhibitor of JNK(c-Jun NH₂-terminal kinase), via interaction with the C-terminus. Itcould also be demonstrated that exogenous (recombinant) GSTP1 proteincould be delivered into macrophages and suppressed iNOS and COX-2expression in cells. Furthermore, intraperitoneally administered GSTP1protein to mice decreased mortality of endotoxic shock significantly andinhibited acute lung injury and peritonitis (Luo et al., Mol. Immunol.46 (2009), 848-857).

EP 1 500 709 A1 suggests GSTP1 as one of 22 inflammation markers.

Various isoforms of GSTP1 have also been found relevant to asthma andchildhood allergic diseases due to the interaction of GSTP1 with airpollution from traffic (and tobacco smoke) in the respiratory system, aswell as association with hypertension in pregnancy (Ohta et al., Sem.Thr. Hem. 29 (2003), 653-659).

Cardiomyopathies (CMP) are a heterogeneous group of diseases, includingthe dilative (DCM), the most frequent entity among cardiomyopathies, andischemic (ICM) forms. Although pharmacotherapy and surgicalinterventions have been improved with survival benefit in CMP, causaltreatment remains to be a vague goal. Therefore, identification ofunknown mechanisms that mediate CMP could be beneficial. Results of thelast decade's work clearly provide evidence that inflammation andenhanced release of pro-inflammatory cytokines, especially tumornecrosis factor (TNF)-α might play a role in the pathogenesis of CMP.However, clinical trials have paradoxically indicated a more complicatedrole for TNF-α in CMP that casted doubts whether TNF-α is a viabletherapeutic target for CMP. The pathology of IHD is immediately relatedto the development of CMP, patients currently receive medications suchas beta-blockers or angiotensin converting enzyme (ACE)-blockers withthe goal to ameliorate the patient's symptoms and to prevent or delaythe development of (ischemic) CMP due to IHD. It is therefore common touse therapies for CMP also for IHD. Importantly, patients who developCMP based on IHD, in particular end-stage CMP, have currently onlylimited therapeutic options (e.g. cardiac transplantation or the use ofventricular assist devices, which can be in turn offered only to alimited number of patients).

There is still an unmet need for further drug targets and treatmentregimen for efficiently preventing and treating CMP and IHD and one ofits major causes, that is ischemic heart diseases (also called coronaryheart diseases that can result in myocardial infarction), especiallyelevated blood pressure-induced CMP and volume-induced CMP.

Cardiomyopathies result in recurrent exacerbation leading tohospitalization or death. Therefore, also close surveillance andthorough clinical tests are required to achieve the optimal tailoredcardiomyopathy treatment in these patients, associated with tremendoushealth care costs. A biomarker that could identify the evolution ofcardiomyopathy and help to optimize treatment monitoring and outcome istherefore highly desirable. Ideally, such a test should be sensitive,specific, none-invasive, quick and cheap. Although, a number ofneurohumoral factors and inflammatory cytokines have been reported asdiagnostic biomarkers in cardiomyopathy, brain-type natriuretic peptide(BNP) and its inactive N-terminal fragment (proBNP) have gained thebroadest acceptance in clinical diagnosis and monitoring of patientswith suspected or established chronic CMP. However, circulating BNP andproBNP levels are dramatically affected by renal function and are age-and gender-dependent. In addition, it remains unknown whether proBNPcould be beneficial in diagnosis of preserved as opposed to reducedejection fraction (EF) CMP.

It is therefore also an object of the present invention to providediagnostic means for cardiomyopathies and ischemic heart diseases.

Therefore, the invention provides Glutathione S-transferase P1 (GSTP1)for the prevention or treatment of cardiomyopathies (CMPs) as well asfor the prevention or treatment of ischemic heart diseases. With thepresent invention it could be surprisingly shown that GSTP1 isbeneficial in CMP treatment and in the treatment of ischemic heartdiseases (IHD). GSTP1 has been described as a relevant protein in cancermedicine (e.g. U.S. Pat. No. 5,427,917 A, U.S. Pat. No. 5,552,277 A andWO 98/21359 A1) and in allergic diseases, such as asthma, especially inconnection with traffic-related air pollution (e.g. Melen et al., Env.Health Persp. 116 (2008), 1077-1084). GSTP1 has also been suggested todecrease mortality of endotoxic shock and inhibition of acute lunginjury and peritonitis (Luo et al., Mol. Immunol. 46 (2009), 848-875).GSTP1 was reported to interact with TRAF2 (tumor necrosis factorreceptor-associated factor 2) as a novel ligand-binding function ofGSTP1 in regulating kinase signalling (Wu et al., Oncogene 25 (2006),5787-5800). Although this provided a new insight for analysing themechanism utilized by GSTP1 to protect cells against different injurystimuli such as cytokines, UV or H₂O₂, and this teaching was regarded asconceivable that GSTP1 functions as a general survival factor by formingGSTP1-TRAF2 complex in tumor cells, inhibition of this complex wasregarded as being limited to antitumor therapy.

According to the present invention it was surprisingly found that GSTP1is specifically upregulated in CMP and IHD patients as compared tocontrols. It was further found that recombinant GSTP1 mediatedGSTP1-TRAF2 association, decreased pro-inflammatory JNK1 and p38activation and TRAF2 expression dependent on its dose and the underlyingform of CMP. This enabled the provision of a new therapy strategy forCMP and IHD by administration of GSTP1. Since GSTP1 amelioratesTNF-α-mediated activation of pro-inflammatory JNK1/p38 by association toTRAF2, GSTP1 is not only beneficial in CMP and IHD treatment but alsofor the prevention of CMP and IHD, especially in risk patients, such ashypertensive patients.

The present invention provides the use of GSTP1 for the manufacture of amedicament for the prevention or treatment of CMP and ischemic heartdiseases. The treatment of CMP and IHD according to the presentinvention involves the administration of an effective amount of GSTP1 toan individual in need of such treatment, e.g. human CMP and IHDpatients. For these reasons and based on the fact that the pathology ofIHD is immediately related to the development of CMP, patients currentlyreceive medications such as beta-blockers or angiotensin convertingenzyme (ACE)-blockers with the goal to ameliorate the patient's symptomsand to prevent or delay the development of (ischemic) CMP due to IHD.The close relationship of the pathology of IHD with CMP that is based onthe remodelling of myocardium in patients with IHD, specially inpatients developing myocardial infarction due to IHD that in turn leadsto remodelling of the cardiac chambers and subsequently to impaired pumpfunction of the heart, a pathological condition that is common to CMPand IHD, specially in myocardial infarction patients, shows theeffectiveness of the present invention for the prevention or treatmentof IHD and CMP. Therefore, it is evident that the present invention isalso suitable for prevention or treatment of IHD. Importantly, patientswho develop CMP based on IHD, in particular end-stage CMP, havecurrently only limited therapeutic options (e.g. cardiac transplantationor the use of ventricular assist devices, which can be in turn offeredonly to a limited number of patients) and this fact constitutes apreferred use of the present invention in order to prevent or treatpatients suffering from IHD, aimed to prevent the development of CMP atthe one hand and to improve or stabilise the clinical condition in IHDpatients on the other.

With the present invention all forms and stages of CMP and IHD, acuteand chronic forms thereof, can be treated, independently of the genesisof the CMP or IHD. Of course, the term “treatment” includespreferentially the amelioration of the disease and the prevention orslowing of progression of the disease. For example, CMPs can be causedby myocardial infarction, can be idiopathic dilative or be caused byhypertension. All these forms can be treated with GSTP1 according to thepresent invention. Specifically ischemic CMPs e.g. caused by myocardialinfarction can be treated with GSTP1, however, also non-ischemic formscan be treated (e.g. idiopathic dilated CMPs or hypertension- orvolume-induced CMPs). Since in all these CMPs, inflammatory pathways areinvolved, this makes them eligible for treatment with GSTP1 according tothe present invention (even rare forms of CMP, if such inflammatorypathways are involved).

Therefore, preferred CMPs according to the present invention are dilatedcardiomyopathy, especially congestive cardomyopathy; obstructivehypertrophic cardiomyopathy, especially hypertrophic subaortic stenosis;other hypertrophic cardiomyopathies, especially nonobstructivehypertrophic cardiomyopathy; endomyocardial (eosinophilic) disease,especially endomyocardial (tropical) fibrosis or Löffler's endocarditis;endocardial fibroelastosis, especially congenital cardiomyopathy; otherrestrictive cardiomyopathies, especially constrictive cardiomyopathy NOS(not otherwise specified (according to ICD-10)); alcoholiccardiomyopathy, cardiomyopathies due to drugs and other external agents,unspecified cardiomyopathies, especially cardiomyopathy (primary)(secondary) NOS; cardiomyopathy in infectious and parasitic diseases,especially cardiomyopathy in diphtheria; cardiomyopathy in metabolicdiseases, especially cardiac amyloidosis; cardiomyopathy in nutritionaldiseases, especially nutritional cardiomyopathy NOS; Gouty tophi ofheart or thyrotoxic heart disease.

Specifically preferred forms of CMP or IHD to be treated (or prevented)according to the present invention are CMPs caused by ischemia,especially by myocardial infarction; by hypertension, or by myocarditis.GSTP1 treatment according to the present invention is thereforepreferably applied in patients with acute myocarditis, preferablyinfective myocarditis, especially septic myocarditis; isolatedmyocarditis, myocarditis in a bacterial disease, especiallydiphtheritic, gonococcal, meningococcal, syphilitic or tuberculousmyocarditis; myocarditis in a viral disease, especially influenzalmyocarditis or mumps myocarditis; acute or chronic myocarditis inChagas' disease, myocarditis in toxoplasmosis, rheumatoid myocarditis orsarcoid myocarditis.

The preferred indications according to the present invention aretherefore those listed under Chapter IX of the ICD-10 (I11-I15,especially I21, I22 and I23; I20-I25, especially I21 and I22; I40-I43,especially I42; I50-I57, especially I50 and I57).

The main focus of the present invention is the treatment of humanpatients; however, based on the present findings, it is clear that alsoanimal (mammal) CMP and IHD in which inflammation is involved can besuccessfully treated by GSTP1 according to the present invention. Thismay be important for farm or zoo animals (especially breeding farmanimals, such as (former) racing horses) or pets, for example dogs, catsand horses.

Although the present invention is also useable for the prevention of CMPand IHD it is clear that the primary focus of the present invention liesin the treatment of human CMP and IHD patients who have already beendiagnosed of CMP or IHD or which have a high risk of developing CMP orIHD, for example patients with hypertension (e.g. Stage 2 patients withsystolic pressure of 160 mm Hg or more and/or with diastolic pressure of100 mm Hg or more). Another preferred embodiment of the preventiveaspect of the present invention is for angina pectoris patients, wherebymyocardial infarction might or might not have occurred in this set ofpatients; and heart insufficiency or heart failure (Mc Murray et al.,Lancet 365 (2005), 1877-1889) may or may not be present. Here, evencontinuous intravenous administration or continuous administration tothe heart muscle is indicated.

Preferred routes of administration of the GSTP1 containing medicamentaccording to the present invention are parenteral routes, preferablyintraperitoneal or intravenous administration, intravenousadministration being specifically preferred. Intravenous administrationcan be performed e.g. via bolus injection or by continuous intravenousdelivery over a longer time period (e.g. 30 min to 6 h, especially 1 to3 h). In the process of heart surgery also administration directly tothe heart (intramyocardial injection) is preferred. With this route,GSTP1 activity can be directly and in high concentrations delivered tothe area of need, e.g. to an infarct region. Another preferred route ofadministration is administration to the coronary sinus (e.g. via acatheter inserted into the coronary sinus). Oral or mucosal routes(although described for GSTP1 in principle; see e.g. U.S. Pat. No.5,976,528 A) are less preferred, since GSTP1 is a protein and for thisroute specific protection measures (enteric coating, encapsulation,etc.) are necessary as well as a significant optimisation with respectto galenic manufacture.

Preferred dosages for administration are dosages of 0.001 to 100 mgGSTP1/kg, preferably 0.01 to 10 mg GSTP1/kg, especially 0.1 to 1 mgGSTP1/kg, to a human individual, preferably via intravenousadministration; or dosages of 0.1 to 10000 U GSTP1/kg, preferably 1 to1000 U GSTP1/kg, especially 10 to 100 U GSTP1/kg, to a human individual,preferably via intravenous administration. These are preferred dailydosages for intravenous application (“kg” means kg body weight of theperson to be treated). During surgery, these dosages may also beapplied, either as a whole or even more than one dosage; or only part ofthe dosage (having in mind that the GSTP1 can directly be applied duringsurgery in the area needed). “Ready-to-use” daily dosage forms (e.g. fora 80 kg and/or a 60 kg human individual) can be pre-manufactured and canbe made storage-stable by lyophilisation or freezing (and keeping ate.g. −20° C.). The dosage can then be fully or only partially consumed(e.g. based on the body weight of the person to be treated).

GSTP1 according to the present invention is also referred to as“GSTP1-1”, “GST class-pi”, “DFN7”, “GST3”, “GST π”, etc. and conjugatesreduced glutathione to a wide number of exogenous and endogenoushydrophobic electrophiles (EC 2.5.1.18; Catalytic activity:RX+glutathione=HX+R—S-glutathione). The human sequence (Seq. ID. No. 1)is listed as “GSTP1_HUMAN, P09211” in the UniProtKB/SwissProt database(HGNC: 4638, Entrez Gene: 2950).

SEQ. ID. NO. 1:   1 mppytvvyfp vrgrcaalrm lladqgqswk eevvtvetwqegslkascly gqlpkfqdgd  61 ltlyqsntil rhlgrtlgly gkdqqeaalv dmvndgvedlrckyisliyt nyeagkddyv 121 kalpgqlkpf etllsqnqgg ktfivgdqis fadynlldlllihevlapgc ldafpllsay 181 vgrlsarpkl kaflaspeyv nlpingngkq

Several variants exist for this human protein (homozygous as well asheterozygous), the most important variants are a Ile to Val exchange atposition 105 and a Ala to Val exchange at position 114 (Melen et al.,2008). Other variants include a Gly to Glu exchange at position 78, aThr to Ser exchange at position 110, a Gly to Glu exchange at position139, a Asp to Tyr exchange at position 147 and a Asp to His exchange atposition 158. Several other SNPs are known without affecting the aminoacid sequence.

If the CMP or IHD patient to be treated with GSTP1 according to thepresent invention has such an allele which results in a (heterozygous orhomozygous) amino acid exchange compared to SEQ.ID.No. 1, it may berecommendable to administer to such a patient the allelic form of thisprotein. This is specifically preferred for patients who are homozygousfor this allele, e.g. patients with homozygous 105Val or 114Val allele(who could then receive the 105Val or 114Val GSTP1 variant,respectively). In such cases, the risk of developing adverse reactions,especially adverse immune reactions (which is always a topic forproteinaceous medicaments), is kept low. Anyway, any ingredients in thepharmaceutical preparation containing GSTP1 according to the presentinvention eliciting or boosting an immune reaction in the patient shouldbe kept at minimum if possible.

Orthologs from mammals are known, e.g. from chimpanzee (Pan troglodytes;NCBI accessions: 745954, XM_001152516.1, XP_001152516.1) and mouse (Musmusculus; NCBI accessions: 148701, NM_013541.11, NP_038569.11,AK0791445, BC0020485), but also from non-mammals, such as zebrafish(Danio rerio) and worm (Caenorhabditis elegans).

The present invention also relates to a pharmaceutical compositioncomprising GSTP1, preferably human recombinant GSTP1 or human placentaGSTP1, and a pharmaceutically acceptable carrier for the prevention ortreatment of cardiomyopathies or ischemic heart diseases.

According to a preferred embodiment, the composition according to thepresent invention contains 1 to 100000 U GSTP1, preferably 10 to 10000 UGSTP1, especially 10 to 1000 U GSTP1. A unit of GSTP1 activityconjugates 1.0 micro-mole of 1-chloro-2,4-dinitrobenzene with reducedglutathione per minute at pH 6.5 at 25° C.

A preferred composition according to the present invention also containsa buffer. Typical buffer systems are the carbonic acid/HCO₃ system (pH6.2 to 8.6; neutral), carbonic acid/silicate buffer (pH 5.0 to 6.2;weakly acidic), acetic acid/acetate buffer (pH 3.7 to 5.7), phosphatebuffer (NaH₂PO₄+Na₂HPO₄; pH 5.4 to 7.8), ammonia buffer (NH₃+H₂O+NH₄Cl;pH 8.2 to 10.2), TRIS/HCl (Tris(hydroxymethyl)-aminomethane; pH 7.2 to9.0), HEPES (4-(2-Hydroxyethyl)-1-piperazinethanesulfonic acid; pH 6.8to 8.2), HEPPS (4-(2-Hydroxyethyl)-piperazine-1-propane sulfonic acid;pH 7.3 to 8.7) or MES (2-(N-Morpholino)ethanesulfonic acid; pH 5.2 to6.7). Preferred buffers according to the present invention is aphosphate buffer, a Tris-HCl buffer or a HEPES buffer, with a pH of 5.5to 9.0, preferably of 6.0 to 8.5, especially of 6.5 to 8.0. Bufferconcentrations can preferably be adjusted to 1 mM to 1 M, especially 10mM to 0.5 M. Other preferred additional ingredients include stabilisers,chelators, salts, etc., such as glycerol, glucose, saccharose, maltose,EDTA or similar substances, NaCl, KCl, NH₄Cl and similar salts usable inpharmaceutical preparations. Of course, all ingredients in apharmaceutical preparation have to be of pharmaceutical grade quality.

Pharmaceutically acceptable carriers preferably used are physiologicalsaline, vegetable oils, mineral oil, aqueous sodium caroboxymethylcellulose or aqueous polyvinylpyrrolidone; however, also sterile watercan be used.

GSTP1 to be used according to the present invention can be provided byvarious ways. As mentioned above, GSTP1 from human placenta is one ofthe preferred natural sources of GSTP1. Other preferred sources arecultures of GSTP1 (over-)expressing cells. GSTP1 preparations are alsocommercially available. The preferred industrial production routeaccording to the present invention is, however, recombinant productionof GSTP1. Recombinant production of GSTP1 is well established, as wellas its purification from such sources (e.g. Luo et al., 2009, Wu et al.,2006; WO 98/21359 A1; U.S. Pat. No. 5,976,528; etc.). Expression vectors(e.g. WO 98/21359 A1, Part D (pages 40 to 51) containing the GSTP1coding sequences are transferred in suitable host cells (such as COS,HEK 293, HeLa, VERO, W138, BHK or MDCK cells, but also yeast, plant,insect and bacterial cells (S. cerevisiae, E. coli, etc.). GSTP1 is thenexpressed and purified according to standard methods. Recombinant GSTP1production may also use variant forms of GSTP1 with additional sequenceswhich facilitate purification, such as metal chelating peptides, proteinA domains, affinity purification tags, etc. (e.g. U.S. Pat. No.5,976,528 A, column 16). Such variants can—after purification or in thecourse of purification—be removed e.g. via cleavable linker sequences(protease, entereokinase, etc.). Purified batch preparations of GSTP1may contain e.g. 1 to 200 U GSTP1 activity per mg total protein.Preferred pharmaceutical GSTP1 compositions contain 5 to 200 U/mgprotein, especially 10 to 150 U/mg protein. The pharmaceuticalpreparation according to the present invention is always provided as anappropriately labelled product which is sterile and useable foradministration to human patients according to the present invention.

According to another aspect, the present invention relates to GSTP1 as adiagnostic target for CMP or IHD. The present invention provides the useof GSTP1 for the diagnosis of CMP or IHD. Although GSTP1 is a known andestablished marker in tumor diagnosis, it was surprising that GSTP1 is abiomarker for CMP or IHD which is even superior to the current “goldstandard”, pro-BNP, with respect to sensitivity and specificity.

The cardiomyopathies or ischemic heart diseases referred to above fortherapeutical use of GSTP1 can also be diagnosed according to thepresent invention.

The present invention therefore relates to a method for diagnosing CMPor IHD, especially the indications mentioned above, by determining theamount of GSTP1 or GSTP1 mRNA in a biological sample and comparing thisamount to the GSTP1 or GSTP1 mRNA amount in a biological sample with adefined and known CMP/IHD status, especially from a healthy humanindividual or a patient. This comparison can be done practically, i.e.in that a real sample is tested in the same manner as the biologicalsample (side-by-side), it can also be a virtual comparison so that thedetermined value is compared with a known value of a healthy or diseasedsample.

Preferred techniques for determining the amount of GSTP1 includeantibody-liked techniques, such as ELISA, optionally combined withelectrophoresis techniques or radio immunoassay (RIA). Electrophoresistechniques may also be combined with MALDI techniques.

Alternatively, GSTP1 mRNA may be determined, e.g. by quantitative realtime RT-PCR.

The preferred diagnosis target is a human patient who is suspected tohave CMP or IHD, a human patient who is at risk for developing CMP orIHD or a human patient who has CMP or IHD (who has to be monitored forprogression of the disease).

Preferred biological samples according to the present invention includehuman blood, plasma or serum samples or human myocardial biopsy samples.These samples can also be tested by routine methods.

Typically, a high concentration of GSTP1 in serum is indicative of CMPor IHD. In healthy persons, serum concentrations of GSTP1 are below 50ng/ml or, preferably, even below 10 ng/ml. A CMP or IHD diseased statuscan be diagnosed at a serum level at ng/ml or above, preferably at 100ng/ml or above. Severe forms of CMP or IHD can be diagnosed at 200 ng/mlor above. Of course, these levels always depend on the technique usedfor assaying the GSTP1 protein. Alternatively, CMP or IHD diseasedstatus can be diagnosed if the serum level of GSTP1 is elevated by atleast the factor 2, preferably by at least the factor 5, compared to ahealthy status, especially the healthy status of the same patient.Severe forms could be diagnosed, if the serum GSTP1 level is elevated byat least the factor 10, especially by at least the factor 20, comparedto a healthy status, especially the healthy status of the same patient.

With the present invention determining the amount of GSTP1 or GSTP1 mRNAin a sample can be performed within a very short time by standard testmethods, such as ELISA or PCR. Based on the DNA and amino acid sequenceaccording to SEQ.ID.NO.2

atgccgccct acaccgtggt ctatttccca gttcgaggcc   60 gctgcgcggc cctgcgcatgctgctggcag atcagggcca gagctggaag gaggaggtgg  120 tgaccgtgga gacgtggcag gagggctcac tcaaagcctc ctgcctatac gggcagctcc  180 ccaagttcca ggacggagac ctcaccctgt accagtccaa taccatcctg cgtcacctgg  240 gccgcaccct tgggctctat gggaaggacc agcaggaggc agccctggtg gacatggtga  300 atgacggcgt ggaggacctccgctgcaaat acgtctccct catctacacc aactatgagg  360 cgggcaagga tgactatgtgaaggcactgc ccgggcaact gaagcctttt gagaccctgc  420 tgtcccagaa ccagggaggcaagaccttca ttgtgggaga ccagatctcc ttcgctgact  480 acaacctgct ggacttgctgctgatccatg aggtcctagc ccctggctgc ctggatgcgt  540 tccccctgct ctcagcatatgtggggcgcc tcagcgcccg gcccaagctc aaggccttcc  600 tggcctcccc tgagtacgtgaacctcccca tcaatggcaa cgggaaacag tga 633and SEQ.ID.NO.1 (and the data base entries mentioned above for the GSTP1gene and the GSTP1 protein), the present invention also provides e.g. aPCR assay for identification of S. aureus infections or a sandwich ELISAassay. In a suitable ELISA according to the present invention, GSTP1 canbe caught out of a biological sample with a GSTP1-specific antibody anddetected and quantified with a second antibody directed against adifferent epitope of GSTP1 or an antibody being specific for theprevious binding event.

Detection of GSTP1 transcripts (m-RNA) is possible by standardquantitative nucleic acid tests, such as quantitative RT-PCR. Thepresent invention therefore also provides a kit for determining theamount of GSTP1 mRNA in a least two synthetic nucleic acid sequences foramplifying at least one nucleic acid sequence encoding GSTP1 or partsthereof, wherein at least of one of the synthetic nucleic acid sequencesin the kit is selected from the group consisting of: (a) a suitableprimer pair, the primers being a nucleic acid sequence comprising 10-30consecutive nucleotides of at least one of the following: (i) the GSTP1mRNA according to SEQ.ID.NO.2 or the complementary sequence thereof; or(ii) from the 5′ or 3′ noncoding region of the GSTP1 gene (e.g. inregions extending up to 500, preferably up to 200, especially up to 100,nucleotides into the 5′- or 3-direction or complementary sequencesthereof; such sequences are present in the sequence library entriesmentioned above); said primers being usable for polymerising anucleotide sequence located between the primers on the GSTP1 gene or its5′- or 3′-region (said polymerizable GSTP1 nucleotide sequence being PCRdetectable and preferably up to 1000 bp, more preferred up to 500 bp,even more preferred up to 300 bp in length; on the other hand, theprimers may also be selected to polymerise (with PCR) the whole GSTP1m-RNA); and (b) suitable reagents for a polymerase chain reaction (PCR).Preferably, the kit further comprises: (c) instructions for use and (d)optionally, a positive and/or negative control for determining and/orquantification of GSTP1 mRNA, especially a control nucleic acid encodingGSTP1 or a fragment thereof. Examples of suitable PCR reagents includePCR reaction buffer, Mg²⁺ (e.g., MgCl₂), dNTPs, DNA polymerases (such asreverse transcriptases and thermostable DNA polymerases (e.g.,Taq-related DNA polymerases and Pfu-related DNA polymerases)), RNase,PCR reaction enhancers or inhibitors, PCR reaction monitoring agents(e.g., double-stranded DNA dye (such as SYBR™ Green), TaqMan™ probes,molecular beacons, and Scorpions™), and PCR-grade water.

The primers described herein are particularly useful in a polymerasechain reaction (PCR) assay. PCR is a practical system for in vitroamplification of a DNA base sequence. For example, a PCR assay may use aheat-stable polymerase and two about 10 to 30 base primers: onecomplementary to the (+)-strand at one end of the sequence to beamplified, and the other complementary to the (−)-strand at the otherend. Because the newly-synthesized biological sample, comprising: (a) aprimer set comprising at DNA strands can subsequently serve asadditional templates for the same primer sequences, successive rounds ofprimer annealing, strand elongation, and dissociation may produce rapidand highly-specific amplification of the desired sequence. PCR also maybe used to detect the existence of a defined sequence in a GSTP1 m-RNAcontaining sample.

By way of example, a typical PCR assay according to the presentinvention might start—optionally after reverse transcription of the mRNAinto DNA—with two synthetic oligonucleotide primers which arespecifically and complementarily binding to two regions of the targetDNA or its complementary strand, respectively, encoding GSTP1 or its 5′-and/or 3′ region (one for each strand) that is to be amplified. Thesemay be added to the target DNA (that needs not be pure) in the presenceof excess deoxynucleotides (dNTPs) and a thermostable DNA polymerase(e.g., Taq polymerase). In a series (typically 20-40) of temperaturecycles, the target DNA may be repeatedly denatured (about 80-100° C.,e.g. 90° C.), annealed to the primers (typically at 40-65° C.), and adaughter strand may be extended from the primers (typically at 65-80°C., e.g. 72° C.). As the daughter strands themselves act as templatesfor subsequent cycles, DNA fragments matching both primers are amplifiedexponentially, rather than linearly. The target DNA needs be neitherpure nor abundant; thus, PCR is specifically suitable in the clinicaldiagnostics according to the present invention.

These tests may preferably employ labels which are also suitable forquantification, such as biotin, fluorescent molecules, radioactivemolecules, chromogenic substrates, chemiluminescence markers, and thelike. The methods for biotinylating nucleic acids are well known in theart, as are methods for introducing fluorescent molecules andradioactive molecules into oligonucleotides and nucleotides. Detectionmethods are well known for fluorescent, radioactive, chemiluminescent,chromogenic labels, as well as other commonly used labels. Briefly,chemiluminescence can be identified and quantitated most directly bytheir emission wavelengths and intensity. When biotin is employed, it isdetected by avidin, streptavidin or the like, which is conjugated to adetectable marker, such as an enzyme (e.g. horseradish peroxidase).Steptavidin binds with high affinity to biotin, unbound streptavidin iswashed away, and the presence of horseradish peroxidase enzyme is thendetected using a luminescence-emission substrate in the presence ofperoxide and appropriate buffers.

Determining the amount of GSTP1 in a sample preferably includes ELISA,RIA and FACS. Detection and quantification of GSTP1 by using anti GSTP1antibodies is well established in the art (especially in tumor medicine)and can be readily applied for the purpose of diagnosing CMP or IHDaccording to the present invention (e.g. WO 98/21359 A1 (“F.Antibodies”); U.S. Pat. No. 5,976,528 A, U.S. Pat. No. 5,427,917 A).Normal or standard values for GSTP1 expression are established bycombining body fluids or cell extracts taken from normal mammaliansubjects, preferably human, with antibody to GSTP1 under conditionssuitable for complex formation. The amount of standard complex formationmay be quantified by various methods, preferably by photometric means.Quantities of GSTP1 expressed in subject, samples, control and disease,from biopsied tissues are compared with the standard values. Deviationbetween standard and subject values establishes the parameters fordiagnosing disease. As already stated above, most preferred samples areof human heart biopsies and samples derived from human blood ofindividual human patients having CMP or IHD, being suspected of havingCMP or IHD or being at risk of developing CMP or IHD.

The present invention is further illustrated by the following examplesand the drawing figures, yet without being restricted thereto.

FIG. 1 shows increased GSTP1 and TRAF2 in DCM and ICM patients. (A)Representative gene array images show increased hybridization to GSTP1and TRAF2 array sequences (arrows) of labelled cDNA probes. (B)Representative protein array images demonstrating enhanced Cy5 (red)staining for cardiac GSTP1 and TRAF2 proteins in DCM and ICM patientscompared to the Cy3 (green) staining of controls. (C) Quantification ofmyocardial GSTP1 and TRAF2 mRNA expressions by real time RT-PCR. GSTP1(*P<0.0001) and TRAF2 (P<0.0001) mRNA expression is significantlyup-regulated in heart failure patients compared to controls. (D)Representative Western blot images and quantification of myocardialGSTP1 and TRAF2 protein expressions corrected for protein loadingcontrol levels. Myocardial protein expression levels were elevated inDCM and ICM for GSTP1 (P<0.0001, P=0.0019) and TRAF2 (P<0.0001, P=0.005)as compared to controls. No significant differences were found in GSTP1mRNA and protein expression when DCM and ICM were compared; althoughTRAF2 mRNA (†P=0.001) and protein (†P0.0001) expression levels weresignificantly higher in DCM as compared to ICM. *, significantlydifferent versus controls.

FIG. 2 shows GSTP1-TRAF2 interaction and activation of JNK and p38 inDCM and ICM. (A) Representative Western blot images and quantificationof myocardial GSTP1-TRAF2 complex formation in DCM, ICM and controls. LVmyocardial lysates were immunoprecipitated (IP) and precipitates wereanalyzed on Western blotts. Significantly lower GSTP1-TRAF2 associationwas found in DCM compared to ICM and controls (*P≦0.008). (B)Representative Western blot images and quantification of myocardialactive JNK1 and p38 protein expression in DCM, ICM and controls. In bothDCM and ICM cardiac protein expression of active JNK as well as p38(P<0.0001) is significantly upregulated as compared to controls.Moreover, DCM patients reveal a significantly higher cardiac JNK and p38protein expression (†P<0.0001) as compared to ICM. All proteinexpressions were corrected for protein loading control levels. *,significantly different versus control.

FIG. 3 shows that GSTP1 modulates cardiac TRAF2-JNK1/p38 system incardiac tissue cultures. (A) Representative Western blot images ofTRAF2, GSTP1, active JNK and active p38 protein expressions in cardiactissue cultures treated with 5 μg/mL recombinant GSTP1. (B)Quantification of TRAF2, active JNKs and active p38 protein expressionsin DCM, ICM and control cardiac tissue cultures treated with recombinantGSTP1 (2.5, 5, 10 μg/ml) for 24 h. Protein expressions of TRAF2 wassignificantly reduced compared to untreated ones dependent on theconcentration used (DCM: P=0.011 for 2.5 μg/mL, P=0.014 for 5 μg/mL,P=0.003 for 10 μg/mL; ICM: P=0.006 for 2.5 μg/mL, P=0.003 for 5 μg/mL,P=0.0001 for 10 μg/mL; controls: P=0.19 for 2.5 μg/mL, P=0.0003 for 5μg/mL, P=0.03 for 10 μg/mL). Following GSTP1 treatment, active JNKexpression declined in DCM, ICM and control cardiac tissue compared tountreated group dependent on the concentration used (DCM: P=0.0009 for2.5 μg/mL, P=0.01 for 5 μg/mL, P=0.0003 for 10 μg/mL; ICM: P=0.0005 for2.5 μg/mL, P=0.007 for 5 μg/mL, P<0.0001 for 10 μg/mL; controls: P=0.001for 2.5 μg/mL, P=0.69 for 5 μg/mL, P=0.71 for 10 μg/mL). Likewise,active p38 expression decreased in response to GSTP1 treatment in DCM,ICM and control cardiac tissue compared to untreated ones dependent onthe concentration used (DCM: P=0.004 for 2.5 μg/mL, P<0.0001 for 5μg/mL, P=0.0009 for 10 μg/mL; ICM: P=0.03 for 2.5 μg/mL, P=0.0001 for 5μg/mL, P=0.008 for 10 μg/mL; controls: P=0.008 for 2.5 μg/mL, P=0.0004for 5 μg/mL and 10 μg/mL). The comparison of the applied GSTP1concentrations with regard to TRAF2, JNK and p38 reduction indicatedthat in DCM and ICM but not controls the differences between 2.5 μg/mLand 5 μg/mL as well as 10 μg/mL were significant. TRAF2: (DCM: P=0.01for 2.5 vs. 5 μg/mL and vs. 10 μg/mL, P=0.62 for 5 vs. 10 μg/mL; ICM:P=0.0007 for 2.5 vs. 5 μg/mL, P=0.0004 for 2.5 vs. 10 μg/mL, P=0.35 for5 vs. 10 μg/mL; control: P=0.07 for 2.5 vs. 5 μg/mL, P=0.06 for 2.5 vs.10 μg/mL, P=0.15 for 5 vs. 10 μg/mL). JNK: (DCM: P=0.0006 for 2.5 vs. 5μg/mL, P=0.003 for 2.5 vs. 10 μg/mL, P=0.48 for 5 vs. 10 μg/mL; ICM:P=0.004 for 2.5 vs. 5 μg/mL, P=0.01 for 2.5 vs. 10 μg/mL, P=0.88 for 5vs. 10 μg/mL; control: P=0.02 for 2.5 vs. 5 μg/mL, P=0.05 for 2.5 vs. 10μg/mL, P=0.97 for 5 vs. 10 μg/mL). p38 (DCM: P=0.0002 for 2.5 vs. 5μg/mL, P=0.04 for 2.5 vs. 10 μg/mL, P=0.76 for 5 vs. 10 μg/mL; ICM:P=0.0006 for 2.5 vs. 5 μg/mL, P=0.02 for 2.5 vs. 10 μg/mL, P=0.41 for 5vs. 10 μg/mL; control: P=0.06 for 2.5 vs. 5 μg/mL, P=0.08 for 2.5 vs. 10μg/mL, P=0.29 for 5 vs. 10 μg/mL). (C) Representative Western blotimages of myocardial GSTP1-TRAF2 complexes from DCM, ICM and controlscardiac tissue cultures treated with recombinant GSTP1 (5 μg/ml) andsubjected to immunoprecipitation (IP). GSTP1-treated cardiac tissuecultures association between GSTP1 and TRAF2 was dose anddisease-independent and significantly higher (P=0.02 for 5 μg/mL GSTP1concentration; results for 2.5 and 10 μg/mL GSTP1 concentration notshown) as compared to untreated samples. *, significantly differentversus control. †, significantly different versus GSTP1 2.5 μg/mltreated tissue cultures. ‡, significantly different versus GSTP1 5 μg/mland 10 μg/ml treated tissue

FIG. 4 shows that TNF-α abrogates sensitivity of TRAF2-JNK1/p38 cascadeto GSTP1 in DCM cardiac cultures. (A) Representative Western blot imagesand quantification (B) of DCM, ICM and control cardiac tissue culturestreated with TNF-α (50 ng/ml) and GSTP1 (5 μg/ml). TNF-α-activated JNK(P=0.08) and p38 (P=0.75) cascade as well as TRAF2 (P=0.18) proteinexpression were not affected by recombinant GSTP1 in. DCM cardiac tissuecultures. In ICM and controls active JNK (P<0.001; P<0.002), active p38(P<0.0002; P<0.0001) and TRAF2 (P<0.001; P=0.003) protein expressionswas markedly reduced in response to GSTP1 stimulation. The comparison ofthe GSTP-treated ICM and DCM cardiac tissue cultures indicatesignificantly lower TRAF2, actives JNK and p38 (†P<0.001) proteinexpressions in ICM as compared to those of DCM. (C) RepresentativeWestern blot images of immunoprecipitated cardiac tissue lysates treatedwith TNF-α (50 ng/ml) and GSTP1 (5 μg/ml). The failure of GSTP1 toassociate with TRAF2 following TNF-α stimulation in DCM cardiac tissuewas present in contrast to ICM and control cardiac tissue.

FIG. 5 shows that higher GSTP1 concentrations rescue TRAF2-mediatedJNK1/p38 downregulation following TNF-α treatment. (A) RepresentativeWestern blot images and quantifications (B) of DCM, ICM and controlcardiac tissue cultures treated with TNF-α (50 ng/ml) and GSTP1 (10μg/ml). (B) The results demonstrate that DCM cardiac tissue culturestreated with GSTP1 at 10 μg/mL and TNF-α express markedly reduced JNKand p38 activation as well as reduced TRAF2 protein expression(*P<0.0001) as compared to TNF-α-treated controls. (C) Theimmunoprecipitates of DCM cardiac tissue lysates demonstrate significant(P≦0.035) increase of GSTP1-TRAF2 association at 10 μg/ml GSTP1 ascompared to 5 mg/ml GSTP1.

FIG. 6 shows that serum and cardiac GSTP1 is associated with CMP. (A)Representative serum protein array images demonstrate enhanced Cy3(green) and Cy5 (red) staining for serum GSTP1 protein in end-stageheart failure (HF) (EF≦35%; n=40) patients compared to controls (EF≧65%;n=40). (B) ELISA measurements indicate significant (*P<0.0001) elevationof serum GSTP1 concentration in end-stage HF patients with (EF≦35%;n=40) versus controls with (EF≧65%; n=40). (C) Representative cardiactissue protein array images demonstrate enhanced Cy3 (green) and Cy5(red) staining for cardiac GSTP1 protein in end-stage HF patients(EF≦35%; n=40) compared to controls (EF≧65%; n=20). (D) RepresentativeWestern blotting images and quantification of myocardial GSTP1 proteinexpression corrected for protein loading control levels. GSTP1 proteinlevels were significantly elevated in end-stage HF patients with EF≦35%versus control cardiac graft tissue (*P≦0.001).

FIG. 7 shows serum GSTP1 and proBNP association with ejection fraction(EF) and the correlation between GSTP1 and proBNP. (A) Heart failurepatients with EF≦22% have significantly higher serum GSTP1concentrations as compared to all other EF groups. Patients withEF33-42%, EF23-32% and EF22% have significantly higher serum GSTP1concentration as compared to those with EF>52% and EF 43-52%. (B)Patients with EF≦22% have significantly higher serum proBNPconcentration as compared to all other EF groups. No significantdifferences in serum proBNP were observed between all other EF groups.(C) Correlation plot of serum GSTP1 and serum proBNP measurements forall study subjects. Significant positive relationship was noted betweenserum GSTP1 and proBNP (r=0.47; P<0.0001). *P<0.001 versus control,†P<0.0001 versus EF>52%, ‡P<0.0001 versus EF 43-52%, §P<0.0001 versus EF33-42%, ∥P<0.0001 versus EF 23-32%.

FIG. 8 shows serum GSTP1 and proBNP correlation to cardiac function. (A)Correlation of serum GSTP1 and proBNP concentrations to cardiac ejectionfraction (EF) in study subjects. The analysis indicates a highersignificant negative correlation of FE with serum GSTP1 (r=−0.74;P<0.0001) as compared to serum proBNP concentration (r=−0.27; P=0.0006).(B) ROC curve analysis indicates at the optimal cut-off level of ≧226ng/ml (black line; AUC=0.891, P<0.0001) a sensitivity of 81% and aspecificity of 82% for serum GSTP1 and at the optimal cut-off level of≧527 pg/ml (red line; AUC=0.624, P=0.0039) a sensitivity of 97% and aspecificity of 26% for serum proBNP to identify EF≦22%. (C) ROC curveanalysis indicates at the optimal cut-off level of ≧76 ng/ml (AUC=0.974,P=0.0008) a sensitivity of 93% and a specificity of 100% for serum GSTP1to identify EF≦42%.

FIG. 9 shows that GSTP1 inhibits the inflammatory cytokines in a ratacute MI model. (A) Immunoprecipitation with Western blotting revealedno differences in GSTP1-TRAF2 complex formation between GSTP1-treatedand control animals; (B) GSTP1 decreased activated JNK1 proteinexpression levels as analyzed by Western blotting; (C) GSTP1 inhibitsthe myocardial tissue mRNA expression of several inflammatory cytokines.*; p<0.01.

FIG. 10 shows that GSTP1 ameliorates inflammation in the rat failingheart. (A) GSTP1-treated animals had significantly higher levels ofcomplex formation between GSTP1-TRAF2, GSTP1-JNK1 and GSTP1-p38, asshown by immunoprecipitation and Western blot analyses; (B) the proteinexpression of activated JNK1, p38 and NF-κB is significantly lower inGSTP1-treated animals as compared to controls as shown by Western blotanalyses; (C) GSTP1 inhibits the mRNA expression of inflammatorycytokines TGF-B, IL-1B, IL-2 and IL-17; and increases the mRNAexpression of anti-inflammatory cytokine IL-10 in failing myocardium ascompared to controls. *; p<0.001.

FIG. 11 shows that GSTP1 attenuates myocardial remodeling in a ratischemia-induced model of heart failure. (A) Goldner trichrome collagenstaining reveals significantly lower tissue remodeling in the heart ofGSTP1-treated animals as compared to controls; (B) left ventricular(infarct) wall thickness is significantly larger in GSTP1-treated ratscompared to controls; (C) representative Tunnel assay images of rathearts treated with GSTP1 or a control vehicle. Bar=25 μm; (D)GSTP1-treated rats had a significantly lower apoptotic index as comparedto controls. *; p<0.01.

FIG. 12 shows that GSTP1 improves the left ventricular function in a ratmodel of ischemia-induced heart failure. (A) GSTP1-treated rats hadsignificantly higher left ventricular ejection fraction at 21 dayspost-myocardial infarct; (B) the left ventricular end diastolic volumeis significantly greater in controls as compared to GSTP1-treatedanimals. *; p<0.05.

EXAMPLES 1.: GSTP1 Ameliorates Inflammation in Cardiomyopathy (CMP) andIschemic Heart Disease (IHD)

Patients and Tissue Sampling

This study was approved by the Ethics Committee of the MedicalUniversity of Vienna. Cardiac tissue samples of 70 CMP patients who gaveinformed consent to be enrolled (idiopathic dilated cardiomyopathy(DCM), n=35; ischemic cardiomyopathy (ICM), n=35) were used for theinitial array analyses. Then, a total of 100 CMP patients (DCM, n=50;ICM, n=50) were included between January 2004 and July 2008. Table 1summarizes the demographic, most important clinical and hemodynamiccharacteristics and the treatment of patients. All patients hadoptimized heart failure treatment and were scheduled for cardiactransplantation according to the American Heart Association criteria.All patients underwent echocardiography, coronary artery angiography,right-heart catheterization and magnetic resonance imaging forevaluation of ventricular function, myocardial viability and standardhemodynamic parameters by independent cardiologists. The case history,clinical test results, and treatment were documented and coded andblinded to the investigators of cardiac biopsies. The control groupconsisted of 20 heart donors with no history of cardiac disease whosehearts could not be transplanted due to quality reasons.

TABLE 1 demographic data, hemodynamic parameters and medication of studypatients. ICM DCM P Male, % 50 48 0.4300 Age (years) 58 ± 6  55 ± 7 0.1521 BMI (kg/m²) 26 ± 3  25 ± 4  0.6688 BSA (m²) 1.9 ± 0.2 1.9 ± 0.20.5883 PAP (mmHg) 30 ± 10 32 ± 8  0.2817 PCWP (mmHg) 21 ± 9  22 ± 7 0.5264 PVR (wood units) 2.5 ± 1.2 2.6 ± 1.7 0.8185 LVEF (%) 19 ± 6  18 ±6  0.3755 Cardiac output (L/min) 4.0 ± 0.8 4.4 ± 1.5 0.1647 Cardiacindex (L/min/m²) 2.1 ± 0.4 2.3 ± 0.8 0.1952 ACE inhibitors (yes/no) 23/424/11 0.1299 Angiotensin II receptor antagonists, % 21 31 0.2494 Betablocker, % 96 83 0.0973 Prostaglandin E2, % 22 20 0.8312 Amiodarone, %26 29 0.8169 Levosimendan, % 19 20 0.6959 ACE, angiotensin convertingenzyme; BMI, body mass index; BSA, body surface area; DCM, dilatedcardiomyopathy; ICM, ischemic cardiomyopathy; LVEF, left ventricularejection fraction; PAP, pulmonary artery pressure; PCWP, pulmonarycapillary wedge pressure; PVR, pulmonary vascular resistance.cDNA Array

Poly(A+)-RNA was isolated with the Oligotex-dT kit (Quiagen, ValenciaCalif.) and first-strand cDNA synthesis was performed with avianmyeloblastosis virus reverse transcriptase (Promega, Madison, Wis.) on 2μg poly(A+) RNA. The RNA strand within the DNA-RNA duplex was degradedand products were purified on a Sephadex G-50 spun-column (Pharmacia,Uppsala, Sweden). For reverse-strand priming, first-strand cDNA was usedto generate [α-32P]dCTP-labeled second-strand cDNA for the cDNA arrays(GEArray Q Human Apoptosis Gene Array, SuperArray Bioscience, Frederick,Md.) as described (Schafer et al., Circulation 108 (2003), 1585-1591).Pooled cDNA hybridization signals were quantified using ImageQuantsoftware (Molecular Dynamics, Sunnyvale, Calif.).

Cardiac Tissue Protein Array

The protein array was performed on antibody Microarray 500 (Clontech,Mountain View, Calif.; 507 proteins) as described (Aharinejad et al.,Circulation 120 (2009a), 11 Suppl:S198-205). The proteins in each samplewere labeled with 2 different fluorescent dyes and incubated onmicroarray antibody-coated slides. The protein fluorescent signals ofboth slides were detected by the GenePix 4000B scanner (MolecularDynamics, Sunnyvale, Calif.). The quantification of the signal wasperformed by calculating an internally normalized ratio (INR), yieldingthe abundance of an antigen in CMP sample relative to that in controlsamples by using the automated Microarray Analysis Workbook(http://bioinfo.clontech.com). Proteins with INR values outside thethreshold interval were considered differentially expressed.

mRNA Isolation and Quantitative Real-Time RT-PCR

Total RNA was isolated from myocardial biopsies using TRIzol(Invitrogen, Carlsbad, Calif.). The tissue was homogenized in a MagNALyser system (Roche, Mannheim, Germany) using MagNA Lyser Green Beadsfor 20-30 seconds at 6000 rpm. Homogenized samples were then mixed with200 μL chloroform, incubated 2-3 minutes at room temperature andcentrifuged at 12000 g for 15 minutes at 4° C. RNA was collected andcDNA was synthesized using 2 μg total RNA and the M-MuLV-RT kit(Fermentas, St. Leon-Rot, Germany). Real-time RT-PCR was performed on aLightCycler instrument (Roche) as described (Aharinejad et al., Am JTransplant. 5 (2005), 2185-2192) The primer sequences weresense/antisense: GSTP1:5′-GGCAACTGAAGCCTTTTGAG-3′/5′-TCATGGATCAGCAGCAAGTC-3′ (SEQ ID NO.3)/(SEQ ID NO.4); TRAF2:5′-GCAGAAGGTCTTGGAGATGG-3′/5′-GGTGGAGCAGCATTAAGGTC-3′ (SEQ ID NO.5)/(SEQ ID NO. 6); and (32-microglobulin:5′-GATGAGTATGCCTGCCGTGTG-3′/5′-CAATCCAAATGCGGCATCT-3′ (SEQ ID NO.7)/(SEQ ID NO. 8). mRNA expression units were determined afternormalization to expression of the housekeeping gene (32-microglobulinas described previously (Aharinejad et al., 2009a and 2005; Pfaffl,Nucleic Acids Res. 29 (2001):e45) and plotted for CMP patients aspercentage relative to those in controls. Measurements were performedthree times. The average value of the three PCR measurements in eachsample was used for data analysis.

Co-Immunoprecipitation and Western Blotting

Human cardiac tissue was lysed in the lysis buffer containing 20 mM Tris(pH 7.5), 135 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mMdithiothreitol (DTT), 25 mM β-glycerophosphate, 2 mM sodiumpyrophosphate, 10% glycerol, 1% Triton X-100, 1 mM sodium orthovanadate,10 mM NaF and 1 mM phenylmethylsulfonyl fluoride (PMSF) supplementedwith complete protease inhibitor cocktail (Roche Applied Science,Indianapolis, Ind., USA) at 4 C as described (Aharinejad et al., 2005).Lysates were centrifuged (15 000 g) at 4 C for 15 min. For identifyGSTP1-TRAF2 complexes formation proteins (500 μg) wereimmunoprecipitated with TRAF2 antibody (0.5 μg, BD Pharmingen, SanDiego, Calif.). The precleared Protein A/G PLUS-agarose beads (SantaCruz Biotechnology) were incubated with immunocomplexes for another 2 hand washed four times with the lysis buffer. The immunoprecipitates werefurther subjected on SDS-PAGE and presided with Western blottinganalysis using anti-GSTP1 monoclonal antibody (Bethyl, Montgomery,Tex.).

Protein lysates (50 μg/lane) were separated by SDS-PAGE (10%) prior toelectrophoretic transfer onto a Nitrocellulose membrane (Bio-Rad,Hercules, Calif.) as described (Aharinejad et al., 2005). The blots wereincubated with primary human monoclonal anti-GSTP1 antibody (Bethyl,Montgomery, Tex.), human monoclonal anti-TRAF2 (BD Pharmingen, SanDiego, Calif.), human polyclonal phospho-p38 (Promega, Madison, Wis.),human polyclonal phosphor-JNK (Santa Cruz Biotechnology, Santa Cruz,Calif.), human polyclonal anti-JNK (Santa Cruz Biotechnology, SantaCruz, Calif.), human polyclonal anti-p38 (Santa Cruz Biotechnology,Santa Cruz, Calif.), prior to incubation with horseradishperoxidase-conjugated secondary antibodies (Amersham Biosciences,Piscataway, N.J.). Protein loading was assessed by Ponceau S stainingand immunodetection was performed by chemiluminescence(Supersignal-West-Pico, Pierce, Rockford, Ill.). Bands were quantifiedby ImageQuant software and specific protein signals were normalized toloading controls and expressed as arbitrary units. The average value ofthree measurements in each sample was used for data analysis.

Cardiac Tissue Culture Experiments

Freshly isolated myocardium of control, DCM or ICM patients (2 to 3 mm³)were incubate (100 pieces per 6-well plate) in DMEM containing 10% fetalcalf serum (Gibco, Carlsbad, Calif.), 50 U/ml penicillin and 250 μg/mlstreptomycin (pH 7.2) at 37° C. in a fully humidified air atmospherecontaining 5% CO₂ as described (Schafer et al., 2003). After 6 hours,wells were treated with recombinant GSTP1 (2.5, 5 or 10 μg/ml; AssayDesign, Ann Arbor, Mich.) and/or TNF-α (50 ng/ml; eBioscience, SanDiego, Calif.) (Wu et al., 2006). Following incubation for 24 hours themedium was changed and cardiac tissue culture lysates were analysed.Experiments were performed in triplicate.

Statistical Analysis

Clinical parameters, patients characteristics, mRNA expression levels ofGSTP1 and TRAF2, and protein expression levels of GSTP1, TRAF2, activep38, and active JNK were compared between groups by χ2 test and one-wayanalysis of variance (one-way ANOVA; Tukey's test) according to thescale of the variable (continuous or categorical). All statisticalanalyses were performed using SAS system for Windows, version 9.1.3 andthe Enterprise Guide, version 4.1 (SAS Institute, Inc., Cary, N.C.).Statistical significance was set at P<0.05. Results are expressed asmean±standard deviation (SD).

Results

GSTP1 and TRAF2 are Overexpressed in Failing Myocardium

The screening tissue profiling arrays identified higher cardiac GSTP1and TRAF2 gene as well as protein expression levels in randomly selectedpatients with both DCM and ICM compared to the control individuals(FIGS. 1A, 1B). To verify the results obtained in screening arrays, themyocardial expression of GSTP1 and TRAF2 was then prospectively examinedby real time RT-PCR and Western blotting in the study cohort. Theseanalyses indicated significantly elevated myocardial mRNA expressionlevels in DCM and ICM for both GSTP1 (P<0.0001) and TRAF2 (P<0.0001) ascompared to controls (FIG. 1C). Likewise, myocardial protein expressionlevels were elevated in DCM and ICM for GSTP1 (P<0.0001, P=0.0019) andTRAF2 (P<0.0001, P=0.005) as compared to controls. (FIG. 1D). Nosignificant differences were found in GSTP1 mRNA and protein expressionwhen DCM and ICM were compared; although TRAF2 mRNA (P=0.001) andprotein (P≦0.0001) expression levels were significantly higher in DCM ascompared to ICM (FIGS. 1C, 1D).

GSTP1-TRAF2 Interaction in JNK/p38 Activation

Since GSTP1 is physically associated with TRAF2 and forms intracellularcomplexes in tumor cells, it was determined whether this pathway isactive in the failing myocardium. The present analyses in cardiac tissuecultures showed that GSTP1 associates with TRAF2 in ICM and controlcardiac tissue, while this association was significantly less (P<0.0001)detectable in DCM (FIG. 2A). As TRAF2 reportedly activates two major MAPKinases, JNK and p38 known to mediate inflammation in failingmyocardium, the myocardial expression of active p38 and JNK protein inthe study patients was examined. The results show that in both DCM andICM cardiac protein expression of active JNK as well as p38 (P<0.0001)is significantly upregulated as compared to controls. Moreover, DCMpatients revealed a significantly higher cardiac JNK and p38 proteinexpression (P<0.0001) as compared to ICM (FIG. 2B). These results showthat GSTP1-TRAF2 association is differentially regulated in DCM and ICMand that this may affect MAPKs activation.

GSTP1 Modulates Cardiac TRAF2-Mediated JNK/p38 Activation

To determine whether GSTP1 modulates TRAF2-mediated JNK and p38activation, cardiac tissue cultures from the failing hearts and controlswere treated with recombinant GSTP1 at three different concentrations.The result show that in GSTP1-treated tissue cultures obtained from DCM,ICM and controls, protein expressions of TRAF2 was significantly reducedcompared to untreated ones. Similarly, following GSTP1 treatment, activeJNK and p38 expression declined in DCM, ICM and control cardiac tissuecompared to untreated group. The comparison of the applied GSTP1concentrations with regard to TRAF2, JNK and p38 reduction show that inDCM and ICM but not controls the differences between 2.5 μg/mL and 5μg/mL as well as 10 μg/mL were significant (FIGS. 3A, 3B).

To test whether exogenously supplemented GSTP1 affects GSTP1-TRAF2complex formation, GSTP1-treated cardiac tissue lysates wereimmunoprecipitated with TRAF2 antibody and immunopellets were subjectedto Western blotting. The results show that in GSTP1-treated cardiactissue cultures association between GSTP1 and TRAF2 was dose anddisease-independent and significantly higher (P=0.07 for 5 μg/mL GSTP1concentration) as compared to untreated samples (FIG. 3C).

These results show that in cardiac tissue cultures GSTP1 affects TRAF2protein expression and acts as a negative regulator of JNK1 and p38activation through GSTP1-TRAF2 complex formation.

TNF-α Abrogates the Sensitivity of TRAF2/JNK/p38 Cascade to GSTP1 in DCM

To determine whether GSTP1 interacts with reported TNF-α-induced JNK andp38 activation in failing myocardium cardiac tissue cultures isolatedfrom DCM, ICM and controls were treated with recombinant TNF-α followedby GSTP1 at 5 μg/ml. These experiments demonstrate that TNF-α-activatedJNK (P=0.08) and p38 (P=0.75) cascade as well as TRAF2 (P=0.18) proteinexpression are not affected by recombinant GSTP1 in DCM cardiac tissuecul tures. However, in cultures isolated from ICM and controls JNK(P<0.001; P<0.002), p38 (P<0.0002; P<0.0001) and TRAF2 (P<0.001;P=0.003) protein expressions are markedly reduced in response to GSTP1stimulation (FIG. 4A). To understand the divergent results obtained forDCM versus ICM group, the cardiac tissue lysates treated with TNF-α andGSTP1 were immunoprecipitated and the immunopellets subjected to Westernblotting. These analyses show failure of GSTP1 to associate with TRAF2following TNF-α stimulation in DCM cardiac tissue (FIG. 4B). Thiseffect, however, was not present in ICM and control cardiac tissue (FIG.4C). These data show the failure of TNF-α-mediated TRAF2 binding toGSTP1 in DCM cardiac tissue cultures at 5 μg/ml GSTP1.

GSTP1 Rescues TRAF2-Mediated JNK1/p38 Downregulation Following TNF-αTreatment at Higher Concentrations

Keeping in mind that TRAF2 cardiac tissue protein expression in DCM wasapproximately two times higher as compared to ICM and controls andconsidering the results described above, we tested the effect of higherGSTP1 concentration following TNF-α treatment. The results demonstratethat DCM cardiac tissue cultures treated with GSTP1 at 10 μg/mL andTNF-α at 50 ng/mL express markedly reduced JNK and p38 activation aswell as reduced TRAF2 protein expression (P<0.0001; FIGS. 5A, 5B).Importantly, the immunoprecipitates of DCM cardiac tissue lysatesdemonstrate significant (P0.035) increase of GSTP1-TRAF2 association at10 μg/ml as compared to 5 mg/ml GSTP1.

These data show that higher GSTP1 concentrations inhibit TNF-α-inducedJNK and p38 activation in DCM through GSTP1-TRAF2 complex forming andalso affect TRAF2 expression.

In the present example GSTP1 and TRAF2 were identified as novelmediators of CMP. Interaction between GSTP1 and TRAF2 has beenpreviously identified in malignancies (Wu et al. 2006), however,connection to CMP has not been suggested before. Interestingly, theability of GSTP1 to associate with TRAF2 differed between DCM and ICM ina TNF-α dependent manner. Although the devastating pro-inflammatory roleof TNF-α signalling in CMP is well established, clinical trials haveparadoxically indicated a more complicated role for TNF-α in CMP.Targeting specific pathways related to TNF-α signalling might thereforeprovide a more selective approach in CMP treatment. TRAF2 is a centralregulator of TNF-α signalling that mediates MAPK activation. At the sametime, GSTP1 can abrogate MAPK activation through association with TRAF2and consequently suppress TNF-α signalling. The present data showsignificant elevation of active JNK and p38 in DCM myocardium comparedto ICM and controls.

Importantly, it was demonstrated that replenishing intracellular storesof GSTP1 with recombinant GSTP1 protein effectively suppresses systemicand localized inflammatory responses through inhibition of MAPKsignalling. Intriguingly, in contrast to the findings observed in nativeDCM myocardium in this study, recombinant GSTP1 induced the inhibitionof JNK and p38 activities as well as decreasing total TRAF2 proteinexpression in cardiac tissue cultures. This effect was accompanied by aconsiderable increase in GSTP1-TRAF2 association in all GSTP1 treatedcardiac cell cultures. Moreover, maximal effects of GSTP1 attenuation onthe TRAF2-JNK/p38 system were observed at 5 μg/ml and were not affectedby further concentration increases. These results contradict thoseobtained with cardiac tissue. However, after GSTP1 treatment, expressionlevels of active JNK/p38 and TRAF2 were highest in cultures from DCMpatients. Since no differences in GSTP1-TRAF2 complex formation uponGSTP1 incubation were observed in DCM compared to ICM and controlcardiac tissue cultures, additional factors could contribute tointracellular regulation of GSTP1-TRAF2 association in CMP. Thediscrepancy between data obtained by analysis of myocardial samples andin vitro experiments could be explained by the presence of a soluble,circulating factor normally present in native myocardium but not incardiac tissue cultures. Since TNF-α is highly elevated in the serum ofCMP patients and has been implicated in the underlying pathology,cardiac tissue cultures were pre-incubated prior to recombinant GSTP1administration in order to introduce the effects of TNF-α in the invitro model. Remarkably, incubation of TNF-α stimulated cardiac tissuecultures with 5 μg/ml GSTP1 did not affect GSTP1-TRAF2 association inICM cardiac cultures whereas in DCM cardiac cultures, GSTP1-TRAF2complex formation was completely abrogated. Moreover, unchanged highexpression of active JNK/p38 as well as TRAF2 was observed exclusivelyin DCM cardiac tissue cultures. These results are in agreement withthose obtained by analysis of myocardial biopsies and fully support thatimpaired GSTP1-TRAF2 interaction in DCM patients resulted in increasedactivity of MAPKs.

These data show, that further increases of GSTP1 concentration restoreGSTP1-TRAF2 association and inhibit JNK1 and p38 activity in DCM cardiactissue cultures upon TNF-α stimulation. These results show that theTNF-α induced attenuation of GSTP1-TRAF2 binding which is solelyassociated with DCM pathology, can be abrogated by higher concentrationsof GSTP1. Moreover, it was demonstrated that in ICM cardiac tissuecultures both JNK1 and p38 activity is inhibited by recombinant GSTP1 ina dose and TNF-α independent manner. These results show that GSTP1action in ICM may occur though a TRAF2-independent manner.

In conclusion, these results show the novel function of GSTP1 inmodulating TNF-α/TRAF2 elicited JNK1/p38 activation in DCM. In addition,GSTP1-TRAF2 association is guarded by TNF-α-stimulation in DCM but notICM or control myocardium in a GSTP1 concentration dependent manner.These findings show (see also: post published abstract Aharinejad etal., Circulation 120 (November 2009), S905; enhancement ofcardiovascular sensitivity to cyclophosphamide: Haberzettl et al.,Circulation 120 (November 2009), S718) the functionality of the presentinvention as a reliable, advanced therapeutic strategy targeting theanti-inflammatory acting of GSTP1.

2.: Serum GSTP1 is a Sensitive Marker of CMP

Patients and Methods

Patients

This study was approved by the Ethics Committee of the MedicalUniversity of Vienna. Serum samples of CMP patients (EF≦35%; n=40) andhealthy volunteers (EF≧65%; n=40) were used for the initial arrayanalyses. Then, a total of 161 patients who gave informed consent to beenrolled were prospectively included. The study cohort comprised 141end-stage CMP patients scheduled for cardiac transplantation and 20patients with preserved EF undergoing conventional isolated aortic ormitral valve surgery. All patients underwent echocardiography, coronaryartery angiography, and right-heart catheterization for evaluation ofventricular function and standard hemodynamic parameters by twoindependent cardiologists. The case history, clinical test results, andtreatment were documented, coded and blinded to the investigators ofserum samples.

The study patients were subdivided in groups based on their leftventricular EF assessed by echocardiography as follows: EF>52%; EF52-43%; EF 42-33%, EF 32-23% and EF≦22% as described (Lee et al.,Circulation 119 (2009) 3070-3077). The control group (n=20) consisted of10 males and 10 females, aged 30-61 years (51.8±3.2 years).

Serum and Cardiac Tissue Collection

Peripheral venous blood samples were collected at CMP diagnosis orshortly before transplant (Aharinejad et al., Am. J. Transplant. 9(2009b), 149-159). Serum samples were coded and stored in liquidnitrogen until analysis. Multiple myocardial biopsies were obtained fromthe anterior left ventricular wall (LV) of the explanted hearts oftransplant patients (EF≦35%; n=40) and from 20 donor hearts that couldnot be transplanted for quality reasons (EF≧65%, mean age 45±10, 12males, 8 females), coded and snap frozen in liquid nitrogen.

Serum and Cardiac Tissue Protein Arrays

For protein array randomly selected serum samples were obtained fromend-stage CMP patients shortly before transplant and from healthyvolunteers and pooled for each group. For cardiac tissue protein arraythe tissue lysates (Abraham et al., Circ. Res. 87 (2000), 644-647) fromLV myocardial biopsies of explanted hearts in transplant patients (n=40)and donor control hearts (n=20) were used. The protein array wasperformed on antibody Microarray 500 (Clontech, Mountain View, Calif.;507 proteins), according to the manufacturer's protocol. The proteins ineach sample were labeled with 2 different fluorescent dyes (Cy3 and Cy5)and incubated on microarray antibody-coated slides. The proteinfluorescent signals of both slides were detected by the GenePix 4000Bscanner (Molecular Dynamics, Sunnyvale, Calif.). The quantification ofthe signal was performed by calculating an internally normalized ratio(TNR) using the automated Microarray Analysis Workbook(http://bioinfo.clontech.com).

GSTP1 and Pro BNP Assays

Enzyme linked immunosorbent assay (ELISA) for GSTP1 (HEPKI™-Pi, BiotrinInternational Ltd., Dublin, Ireland) was performed according to themanufacturer's protocol. The substrate reaction was quantifiedspectrophotometrically by using a 96-well automated microplate reader(Anthos, Salzburg, Austria) at 450 nm. N-terminal proBNP was measured inundiluted serum automatically by a chemiluminescent noncompetitive ELISA(Roche Diagnostics, Indianapolis, Ind.) on a Roche Elecsys 2010analyzer.

mRNA Isolation and Quantitative Real-Time RT-PCR

Total RNA was isolated from LV biopsies using TRIzol (Invitrogen,Carlsbad, Calif.) with a MagNA Lyser system (Roche, Mannheim, Germany).Real-time RT-PCR was performed on a LightCycler instrument (Roche) asdescribed (Aharinejad et al., 2009b, Abraham et al, 2000). The primersequences were sense/antisense: GSTP1:5′-CCAAAGGTGGTGAGCTTCAT-3′/5′-TCTACCCAGCATGGAGGAAC-3′ (SEQ ID NO.9)/(SEQ ID NO. 10); and (32-microglobulin:5′-GATGAGTATGCCTGCCGTGTG-3′/5′-CAATCCAAATGCGGCATCT-3′ (SEQ ID NO.7)/(SEQ NO ID. 8). mRNA expression levels of GSTP1 was normalized to the(32-microglobulin signal as a housekeeping gene (Aharinejad et al.,2009b, Abraham et al, 2000). The average value of three PCR measurementsin each sample was used for data analysis.

Western Blotting Analysis

Tissue lysates were prepared (Aharinejad et al., 2009b) and GSTP1expression was analyzed by Western blotting using primary humanmonoclonal anti-GSTP1 antibody (Bethyl, Montgomery, Tex.). Protein bandswere quantified by ImageQuant software and specific protein signals werenormalized to loading controls. The average value of three measurementsin each sample was used for data analysis.

Statistical Analysis

GSTP1 and proBNP serum concentrations were compared between patientgroups by analysis of variance (one-way ANOVA; Tukey's test). Toinvestigate the relationship between GSTP1 and proBNP, as well as therelationship of GSTP1 to age, PAP, PCWP, PVR, cardiac index andcreatinine, Spearman's rank correlation coefficients (rS) were computed.Univariate logistic regression was used to describe the utility of GSTP1and proBNP as predictors of EF. The corresponding receiver operatingcharacteristic curve (ROC) analyses were used to find optimal cut-offlevels. Sensitivity and specificity of GSTP1 and proBNP cut-offs werecalculated by table analysis. All statistical analyses were performedusing SAS system for Windows, version 9.1.3 and the Enterprise Guide,version 4.1 (SAS Institute, Inc., Cary, N.C.). Statistical significancewas set at P<0.05. Results are expressed as mean±standard deviation.

Results

GSTP1 is Associated with CMP

The demographic data, clinical characteristics and the most CMP-relevantmedication of the study patients are shown in Table 2.

TABLE 2 Demographic data, clinical characteristics and the mostCMP-relevant medication of the study patients. Value Characteristic Age(years) 53 ± 13 Male/Female  119/42 (74%/26%) Diabetes mellitus 34 (21%)IDDM 22 (14%) NIDDM 12 (7%)  NYHA Functional Classification II 25 (15%)III 94 (59%) IV 42 (26%) Diagnosis Valvular Disease 20 (12%) ICM 43(27%) DCM 86 (53%) Others 12 (8%)  Hemodynamic parameters EF (%) 24 ± 14PAP (mmHg) 31 ± 9  PCWP (mmHg) 21 ± 8  PVR (Wood units) 2.58 ± 1.41Cardiac Index (l/min/m²) 2.20 ± 0.64 Laboratory findings Creatinine(mg/dl) 1.32 ± 0.44 GSTP1 (ng/ml) 271 ± 173 proBNP (pg/ml) 1200 ± 630 Medication ACE blocker 111 (69%)  Angiotensin II receptor antagonists 39(24%) Beta blocker 131 (81%)  Levosimendan (S_(IMDAX′)) 21 (13%) DCM =dilated cardiomyopathy; EF = left ventricular ejection fraction; ICM =ischemic cardiomyopathy; IDDM = insulin-dependent diabetes mellitus;NIDDM = non-insulin-dependent diabetes mellitus; NYHA = New York Heartassociation; PAP = pulmonary artery pressure; PCWP = pulmonary capillarywedge pressure; PVR = pulmonary vascular resistance.

In the targeted protein array screen of pooled serum samples in selectedCMP patients GSTP1 was identified as a novel protein Lo be associatedwith CMP. FIG. 6A shows the array images of GSTP1 and indicates that itsserum protein levels are increased in CMP patients as compared tohealthy volunteers. Following these screening analyses, serum GSTP1concentration was determined in the same patient cohort selected forscreening analyses by GSTP1-specific ELISA. These results show thatserum GSTP1 concentrations were significantly upregulated in end-stageHF patients as compared to the control samples (FIG. 6B, P≦0.001).

To learn about the myocardial GSTP1 protein levels, a tissue proteinprofiling was initiated using the LV myocardial samples of the sameend-stage HF patients analysed by serum arrays and used LV myocardialbiopsies of donated hearts as controls. The results of tissue profilingindicate elevated GSTP1 expression levels in myocardium of end-stage HFpatients as compared to controls (FIG. 6C). To validate these findings,Western blotting analyses were performed on cardiac tissues and foundsignificantly upregulated GSTP1 protein expression levels in patientswith end-stage HF as compared to controls (P<0.001; FIG. 6D).

Association of GSTP1 and proBNP with EF

To understand the association of GSTP1 with CMP, its serum levels wereanalyzed in all study patients and plotted these results in relation tothe patient's EF. By selecting this type of analysis that enablesdifferentiation between preserved and reduced EF, significantly higherserum GSTP1 concentrations were found in patients with EF≦22% ascompared to all other EF groups (FIG. 7A; P<0.0001). In addition, CMPpatients with an EF between 23-32% and EF 33-42% had significantlyhigher GSTP1 serum concentrations as compared to those with an EFbetween 43-52% or >52% (FIG. 7A; P<0.0001). The same type of analysisfor serum proBNP revealed that in the present patient cohort only thosewith EF≦22% had significantly higher serum proBNP concentrations(P<0.0001) as compared to all other EF groups except for patients withan EF between 33-42% (FIG. 7B). No significant differences were observedfor circulating proBNP when other BF groups were compared. In the entirepopulation, a significant positive relationship was noted between GSTP1and proBNP (r=0.47, P<0.0001; FIG. 7C).

Diagnostic Value of Serum GSTP1 as Compared to proBNP in CMP

To illustrate the correlation between serum GSTP1 and proBNP with EF askater plot analysis was performed including all study patients (FIG.8A). These analyses revealed a significant negative correlation(r=−0.74; P<0.0001) between GSTP1 and EF which was higher than thecorrelation between proBNP and EF (r=−0.27; P=0.0006). When entering theserum GSTP1 measurements in ROC curve analysis for the EF≦22%, which wasidentified to be significantly different for both GSTP1 and proBNP inunivariate logistic regression analyses, at an optimal cut-off level of≧226 ng/ml serum GSTP1 had a sensitivity of 81% and a specificity of 82%to identify EF≦22% (AUC=0.891, P<0.0001). The same type of analysis at acut-off level of ≧527 pg/ml revealed a sensitivity of 97% and aspecificity of 26% for serum proBNP to diagnose CMP patients with EF≦22%(AUC=0.624, P=0.0039) (FIG. 8B). Then the ability of GSTP1 serum levelsto diagnose higher EF patient groups was analyzed, as indicated by theunivariate analyses shown above (proBNP was not significantly differentbetween patients groups with EF>22%). The ROC curve analyses indicatedthat GSTP1 at an optimal cut-off level of ≧76 ng/ml diagnoses CMPpatients with EF≦42% with a 93% sensitivity and a 100% specificity(AUC=0.974, P=0.0008) (FIG. 8C).

Correlation of GSTP1 and proBNP with Demographic and Clinical Parameters

The associations between GSTP1 and proBNP and clinical characteristicsof the study patients is illustrated in Table 3.

TABLE 3 Spearman correlation analysis between GSTP1 and proBNP andclinical variables of study patients GSTP proBNP Covariates r_(s) Pr_(s) P GSTP1 ng/ml 0.4690 <0.0001 EF −0.7442 <0.0001 −0.2666 0.0006 Age(years) −0.1375 0.0241 0.1756 0.0259 PCWP (mmHg) 0.0577 0.5027 −0.04510.6039 PVR (Wood units) −0.0620 0.4682 −0.0594 0.4908 Cardiac Index−0.1103 0.2534 0.1531 0.1153 (l/min/m²) Creatinine (mg/dl) −0.15600.0686 −0.0649 0.4543 EF = ejection fraction; PAP = pulmonary arterypressure; PCWP = pulmonary capillary wedge pressure; PVR = pulmonaryvascular resistance.

For both serum GSTP1 (r=−0.137; P=0.0241) and proBNP (r=0.175; P=0.0259)only a slightly but not relevant correlation with patient age was foundamong the study patients. No significant correlation between serum GSTP1concentration and pulmonary artery pressure, pulmonary capillary wedgepressure, pulmonary vascular resistance, serum creatinine level andcardiac index was observed. Likewise, no association to gender anddiabetes mellitus (P≧0.408) was observed for both serum GSTP1 andproBNP.

Significant treatment gaps in the use of evidence-based managementpersist despite optimistic results from randomized clinical trials. Theemergence of cardiac biomarkers as increasingly effectual clinical toolssuggests the potential to successfully guide therapy and reduce diseaseburden. Innovative studies have identified a variety of novel molecularmarkers of diagnostic and prognostic value in CMP. ProBNP is currentlyaccepted as a surrogate parameter in CMP monitoring, yet the clinicalroutine work indicates divergent results in CMP patients. The purpose ofthe present study was therefore to find a non-invasive and rapidlyresponding CMP serum marker that allows monitoring of CMP patients withboth reduced and preserved EF. The targeted screening according to thepresent invention showed that GSTP1 is associated with end-stage CMP.Serum GSTP1 concentrations specifically diagnosed CMP with significantassociation to EF independently from demographical and clinicalcharacteristics in our patient cohort. Of note, markedly highercorrelation coefficients were shown for serum GSTP1 association to EF ascompared to proBNP. In addition, serum GSTP1 shows better diagnosticpower in CMP patients with EF≦22% as compared to proBNP. Moreimportantly, GSTP1 diagnosed EF≦42% whereas proBNP failed.

It is well established that EF is a determinant of cardiac risk in CMPpatients. The hazard ratio for all-cause mortality increased by 39% forevery 10% reduction in EF below 45%. However, CMP clinical features canoccur in patients with EF>45% referred to as CMP with preserved EF. Thusthe present finding that GSTP1 is able to discriminate CMP patients withEF≦42% is of important diagnostic and prognostic significance. AlthoughproBNP is an established tool for CMP diagnosis and correlates to EF thereported values regarding proBNP are quite divergent and are evidentlyelevated in both CMP with preserved EF and reduced EF resulting in itslimited clinical use. On the other hand proBNP is recommended to bemainly used for exclusion of CMP with normal EF in patients withsymptoms attributed to CMP. However, given that gender and older age areassociated with higher proBNP levels (Costello-Boerrigter er al., J. Am.Coll. Cardiol. 47 (2006), 345-353), the proposed diagnostic propertiesof proBNP might be too unspecific to differentiate CMP with preserved EFin elderly patients.

Reportedly, proBNP can predict EF<30% with a sensitivity and specificityof 90% and 71%, respectively in a CMP population with EF<45%. Otherstudies reported that plasma proBNP detect EF<28% with 77% sensitivityand 69% specificity in a patients cohort with EF<50%, and it was foundthat proBNP can predict EF<40% with an area under the curve of 0.69. Inthe present study proBNP had 97% sensitivity but only 26% specificity todiagnose EF≦22% with an area under the curve of 0.62. The lowerspecificity of proBNP in the present study could be explained by thefact that our cut-off level was selected to identify lower EFs ascompared to other studies. This criterion was selected to demonstrateutility of GSTP1 for diagnosing EF≦22%. Moreover, the present study hadno exclusion criteria for EF and this could have influenced the testcharacteristics regarding proBNP that was previously shown to vary inCMP patients with preserved EF.

The reason for serum GSTP1 rising in CMP patients is unclear. However,evidence is accumulating that GSTP1 participates in regulation of stresssignalling and protects cells against apoptosis via its non-catalytic,ligand-binding activity.

The results of the present study show that GSTP1 is a sensitive,specific, cheap and quickly measurable serum marker superior to proBNPin CMP. Importantly, GSTP1 discriminates between preserved and reducedEF with a high sensitivity and specificity and can therefore serve as anovel tool in guiding of clinical trials in CMP patients.

3.: Animal IHD and CMP Model for GSTP1 Treatment

In order to have a model that mimics both IHD and CMP in humans, themodel of ligation of the anterior branch of the left coronary artery inthe rat has been selected to further prove the principle of the presentinvention. In this model, IHD is induced following the ligation of thesaid coronary artery branch, and over time the animals will subsequentlydevelop CMP. Therefore, the model is best suited to show the efficacy ofthe suggested invention for prevention or treatment of both IHD and CMP.

The following experiments will be performed according to the publishedprotocol by Aharinejad et al. (Cardiovasc. Res. 79 (2008), 395-404). Theinvestigation will conform to the Guide for the Care and Use ofLaboratory Animals published by the US National Institutes of Health(NIH Publication No. 85-23, revised 1996) and will be confirmed by theInstitutional Ethics Committee at the Medical University of Vienna.Prof. Dr. Seyedhossein Aharinejad, MD, PhD will be in charge of thedesign and supervision of performing the study.

Male, Sprague Dawley rats (Harlan, Borchen, Germany) will be coded forexperiments. Due to expected hemodynamic compromise, ventricularfibrillation, myocardial rupture and bleeding following myocardialinfarction (MI), the number of surviving animals is expected to be about60-70%. Therefore, in each group with myocardial infarction, a total of18 animals will be included. Rats will be anesthetized withintraperitoneal injection of Ketasol (100 mg/kg body weight) and Rompun(10 mg/kg body weight), intubated tracheally using a 14-gauge catheterand ventilated with 1.5% Isofluran at 55 cycles per minute (tidalvolume: 2.5 ml) before undergoing a left lateral thoracotomy. Theanterior branch of the left coronary artery (LCA) will be either ligatedwith a 7-0 polypropylene snare (Ethicon, Somerville, N.J.) or leftintact in sham procedure. At day (d) 7 post MI, left ventricular (LV)function will be assessed by echocardiography. Then, animals withcomparable base-line cardiac function will be coded and assigned togroups 1-8 as shown in Table 4. To treat the expected pain following MI,animals will receive immediately after MI induction and on the first daypost MI 0.6 mg/100 g body weight Piritramid (Dipidolor^(c) in 5% glucosesolution) s.c. Further, animals will receive Piritramid via their drinkwater for 3 days at 0.6 mg/100 g body weight (250 ml water plus 20 ml 5%glucose solution) and at days 4-7 post MI at 0.3 mg/100 g body weight.

On d7, immediately after MI induction, treatment will start in groups1-4 as follows. Group 1 will receive daily intraperitoneal injections ofRinger's solution for two weeks (control), while groups 2-4 will receivedaily injections of recombinant GSTP1 at a dose of 10, 20 and 40 mg/bodyweight for two weeks, respectively. On d21 the treatment will start ingroups 5-8 as follows. Group 5 will receive daily intraperitonealinjections of Ringer's solution for two weeks (control), while groups6-8 will receive daily injections of recombinant GSTP1 at a dose of 10,20 and 40 mg/body weight for two weeks, respectively. Group 9 includes10 animals with sham procedure. On d52 and d86, the LV function will bere-assessed, and animals will be sacrificed on day 88 post MI.

TABLE 4 Groups of animals and their treatment No. of Application andGroup Treatment Sacrifice animals dose 1 Ringer d88 18 i.p. 2 GSTP1 d8818 i.p., 10 mg/kg bw, 2 weeks 3 GSTP1 d88 18 i.p., 20 mg/kg bw, 2 weeks4 GSTP1 d88 18 i.p., 40 mg/kg bw, 2 weeks 5 Ringer d88 18 i.p. 6 GSTP1d88 18 i.p., 10 mg/kg bw, 2 weeks 7 GSTP1 d88 18 i.p., 20 mg/kg bw, 2weeks 8 GSTP1 d88 18 i.p., 40 mg/kg bw, 2 weeks 9 Sham d88 10 — TotalNo. of animals 154  1 Ringer d88 18 i.p. 2 GSTP1 d88 18 i.p., 10 mg/kgbw, 2 weeks 3 GSTP1 d88 18 i.p., 20 mg/kg bw, 2 weeks 4 GSTP1 d88 18i.p., 40 mg/kg bw, 2 weeks 5 Ringer d88 18 i.p. 6 GSTP1 d88 18 i.p., 10mg/kg bw, 2 weeks 7 GSTP1 d88 18 i.p., 20 mg/kg bw, 2 weeks 8 GSTP1 d8818 i.p., 40 mg/kg bw, 2 weeks 9 Sham d88 10 Total No. of animals 154 bw: body weight

The following parameters will be evaluated (1-11):

-   1. LV ejection fraction (EF), LV end systolic volume (ESV), LV end    diastolic volume (EDV) using echocardiography-   2. Heart-to-body weight ratio-   3. Histology and immunocytochemistry for infarct size, infarct wall    thickness and fibrosis as well as angiogenesis, inflammatory    proteins including p38, june kinase and interleukins (only examples    are mentioned for inflammatory pathways)-   4. Signaling pathways-   5. Promoter binding assays-   6. TUNEL-assay for apoptosis evaluation-   7. Quantitative RT-PCR as well as Western Blotting, and    immunoprecipitation for evaluation of GSTP1 and related to its    pathways, TRAFs, TNF-α and its receptors; MMPs and their inhibitors    TIMPs, VEGF-A and its receptors (only examples are mentioned here)-   8. ELISA of serum GSTP1 levels and related proteins

All data will be coded and stored by using Microsoft ACCESS. Analysis ofvariance (ANOVA; t-Test or non-parametric ANOVA) and chi2 test will beused based on dependence of the parameter to be analyzed (numeric oralphanumeric, normal distribution or not) to compare the data betweenthe groups. The Spearmans' correlation test and logistic regressionanalysis will be used to assess the correlation between the evaluatedparameters. SAS version 9.1.3 will be used for statistical analyses.

4.: Effect of GSTP1 Treatment on Inflammation, Cardial Morphology andFunction in a Rat Model of Acute Myocardial Infarction andIschemic-Induced Heart Failure

The following experiment proves the principle, whether glutathioneS-transferase P1-1 (GSTP1) treatment is beneficial in counteracting theinflammatory cascades activated following acute myocardial infarctionand in the process of the resulting ischemia-induced heart failure in anaccepted experimental rat model.

Materials and Methods

Animals and Myocardial Infarction Model

All experiments were approved by the Institutional Animal Care and UseCommittee at the Vienna Medical University. A total number of 60 maleSprague Dawley rats (Harlan, Borchen, Germany) were coded forexperiments. Due to hemodynamic compromise, ventricular fibrillation,myocardial rupture and bleeding following myocardial infarction, thenumber of surviving animals with comparable base-line cardiac functionwas 48. Rats were anaesthetized, intubated tracheally and ventilatedwith an oxygen/isoflurane mixture before undergoing a left lateralthoracotomy. The branch of the left coronary artery was then ligatedwith a 7-0 polypropylene snare (Ethicon, Somerville, N.J., USA). Anintraperitoneal single dose injection of either recombinant GSTP1 (1mg/kg dissolved in 1 ml phosphate-buffered saline (PBS); n=24) (AssayDesign, Ann Arbor, Mich.) or 1 ml PBS (n=24) was applied 1 h afterinduction of myocadial infarction. The left ventricular (LV) functionwas assessed by echocardiography one day (n=48) and three weeks (n=24)after induction of myocardial infarction as described below. A total of12 animals in each group were then sacrificed one day after treatment;and the remaining 12 animals in each group were sacrificed 3 weeks afterinduction of myocardial infarction (Aharinejad et al., 2008).

Echocardiography Hemodynamic Measurements

In anesthetized rats, maximal LV long-axis lengths (L) and endocardialarea tracing were measured using a 15 MHz linear array scan head tocalculate LV end-diastolic (LVEDV) and endsystolic (LVESV) volume andejection fraction (LVEF=LVEDV−LVESV/LVEDV). Measurements utilized threeconsecutive cardiac cycles (Aharinejad et al., 2008).

Histology and Immunocytochemistry

The ventricles were cross-sectioned at the midpoint of their long axis,before they were either frozen or immersion-fixed in formalin.Paraffin-embedded specimens were used for H&E, Tunnel assay as well asGoldner trichrome collagen staining. TUNEL assays (In situ Cell DeathDetection Kit) were performed according to the manufacturer's protocol(Roche Molecular Biochemicals, Basel, Switzerland) in triplicate. Slideswere counterstained with 4,6-diamidino-2-phenylindole (DAPI, MolecularProbes, Eugene, Oreg.) and embedded in AF1 antifadent (Citifluor,Leicester, UK). Digital images were obtained by fluorescence microscopy(Nikon, Melville, N.Y.). Morphometry was carried out as described(Aharinejad et al., 2008).

mRNA Isolation and Quantitative Real-Time RT-PCR

Total RNA was isolated from myocardial tissues using TRIzol (Invitrogen,Carlsbad, Calif.) and MagNA Lyser system (Roche, Mannheim, Germany) asdescribed (Aharinejad et al., 2008). Real-time RT-PCR measurements intriplicates were performed on a LightCycler instrument (Roche) asdescribed (Aharinejad et al., 2008). The primer sequences weresense/antisense: GSTP1:5′-GGCAACTGAAGCCTTTTGAG-3′/5′-TCATGGATCAGCAGCAAGTC-3′ (SEQ ID NO.3)/(SEQ ID NO. 4); tumor necrosis factor receptor-associated factor 2(TRAF2): 5′-GCAGAAGGTCTTGGAGATGG-3′/5′-GGTGGAGCAGCATTAAGGTC-3′ (SEQ IDNO. 5)/(SEQ ID NO. 6); and (32-microglobulin:5′-GATGAGTATGCCTGCCGTGTG-3′/5′-CAATCCAAATGCGGCATCT-3′ (SEQ ID NO.7)/(SEQ ID NO. 8). mRNA expression were calculated and plotted fortreated animals as percentage relative to that in controls.

Co-Immunoprecipitation and Western Blotting

Cardiac tissue lysates were prepared as described (Schafer et al.,2003). For GSTP1-TRAF2, GSTP1-JNK1 and GSTP1-p38 complex detection,proteins (500 μg) were immunoprecipitated with 0.5 μg of correspondingantibodies against TRAF2 (BD Pharmingen, San Diego, Calif.), JNK1 andp38 (Santa Cruz Biotechnology, Santa Cruz, Calif.). The immunocomplexeswere incubated with A/G PLUS-agarose beads (Santa Cruz Biotechnology)and precipitated by Western blotting using GSTP1 antibody (Bethyl,Montgomery, Tex.) as described (Schafer et al., 2003). The blots wereincubated with primary human monoclonal GSTP1 (Bethyl) and TRAF2 (BDPharmingen) or polyclonal phospho-p38 (Promega, Madison, Wis.),phospho-JNK1, JNK1 and p38 (Santa Cruz Biotechnology) antibodies priorto incubation with secondary antibodies (Amersham Biosciences,Piscataway, N.J.). All experiments were performed in triplicate andexpression levels were corrected for control loading protein.

Statistical Analysis

All parameter were compared between groups by χ² test and one-wayanalysis of variance (one-way ANOVA; Tukey's test) according to thescale of the variable (continuous or categorical). All statisticalanalyses were performed using SAS system for Windows, version 9.1.3 andthe Enterprise Guide, version 4.1 (SAS Institute, Inc., Cary, N.C.).Statistical significance was set at P<0.05. Results are expressed asmean±standard deviation (SD).

Results

GSTP1 Ameliorates the Myocardial Overexpression of InflammatoryCytokines Following Acute MI

Immunoprecipitation and Western blot analyses revealed that in the acutemyocardial infarction model, GSTP1 treatment did not change theformation of GSTP1-TRAF2 complexes (FIG. 9A). Western blotting revealedthat although GSTP1 could reduce the cardiac tissue protein levels ofactivated JNK1 (p<0.01; FIG. 9B), there were no difference in theprotein expression levels of activated P38 and NF-kB as compared tocontrols. Moreover, GSTP1 inhibited the cardiac tissue expressions ofinflammatory cytokines including TGF-β, IL-1β, IL-2 and IL-17 (p<0.01;FIG. 9C) as compared to controls; although IL-10 expression levels didnot significantly change following GSTP1 treatment (FIG. 9C).

GSTP1 Ameliorates the Myocardial Expression of Pro-InflammatoryCytokines Through TRAF2-Regulated NF-kB Signaling in the FailingMyocardium

Immunoprecipitation and Western blot analyses revealed thatGSTP1-treated rats had significantly increased formation of GSTP1-TRAF2,GSTP1-JNK1 and GSTP1-p38 complexes in their failing cardiac tissue at 3weeks post-MI as compared to controls (p<0.001; FIG. 10A). In addition,GSTP1-treated rats had significantly reduced protein expression ofactivated JNK1 and p38 in their failing myocardial tissue as compared tocontrols (p<0.001; FIG. 10B). Moreover, GSTP1-treated rats had lowercardiac tissue mRNA expressions of TGF-β, IL-1β, IL-2 and IL-17 ascompared to controls (P0.001; FIG. 10C). In contrast, cardiac tissuemRNA expression of the anti-inflammatory cytokine IL-10 wassignificantly increased in GSTP1-treated animals as compared to controls(p<0.001; FIG. 10C). These results show that GSTP1 treatment inhibitsTRAF2-mediated JNK1, p38 and NF-kB pro-inflammatory signaling andameliorates myocardial cytokine expression in ischemia-induced heartfailure.

GSTP1 Ameliorates Remodeling in Rat Failing Myocardium

The morphometric analyses revealed that LV wall thickness was increasedsignificantly in GSTP1-treated (2.2±0.4 mm) as compared to control rats(1.3±0.5 mm; FIGS. 11A and 11B). To analyze the effect of GSTP1treatment on rescuing myocardial tissue in the infarct and peri-infarctareas, TUNEL assay was performed. The results of these assays showsignificant reduction of apoptotic events in the infarct andperi-infarct area of GSTP1-treated as compared to control animals (FIGS.11C and 11D). These data show that GSTP1 treatment significantly reducesmyocardial remodeling and post-MI myocardial tissue apoptosis in the ratfailing heart.

GSTP1 Improves Cardiac Function

Having shown that GSTP1 is effective in counteracting inflammation andmyocardial remodeling in a rat model of ischemia-induced heart failureand acute myocardial infarction, the LV function in the failing ratmyocardium was proven. The results showed that left ventricular ejectionfraction (LVEF) at 3 weeks post-MI was significantly higher in GSTP1treated rats as compared to untreated group (p<0.05, FIG. 12A).Moreover, LV end-diastolic volume (LVEDV) was improved (decreased)significantly in the GSTP1 treated group at 3 weeks post-MI compared tocontrol animals (p<0.05, FIG. 12B). These data show that GSTP1 treatmentsignificantly improves LVEF and attenuates LV dilation associated withischemia-induced heart failure.

The role of the inflammatory mediators in the pathophysiology of heartfailure has gained a growing interest over the last two decades.Different experimental studies have described the negative inotropiceffect of TNF-α on the LV function. Moreover, it has been reported thatTNF-α promotes LV remodeling, cardiac hypertrophy and progressivecardiomyocytes loss through apoptosis. The growing evidence on thepathological role of inflammatory mediators in the setting of heartfailure have resulted in a series of multicenter clinical trials thatintended to target TNF-α in heart failure patients. However, the resultsof these trials were discouraging. One explanation of these results isthat the low physiological levels of TNF-α might be important forcardiac tissue remodeling and repair. In concert with this argument,experimental studies have described an opposing role of TNF-α in cardiactissue remodeling through its two different receptors TNFR1 and TNFR2.It has been shown that while TNFR1 aggravates remodeling, hypertrophyand apoptosis, TNFR2 ameliorates these events. However, the mechanism ofTNFR2-mediated effect in heart failure remains unclear. Recently, it hasbeen shown that TRAF2 is a central regulator of TNF-α signaling thatmediates MAPK activation. Moreover, the interaction between TNFR2 andTRAF2 results in activation of NF-κB. Furthermore, it has been reportedthat GSTP1 is an important negative regulator of TNF-α-induced signalingby forming interactions with TRAF2. In line with this, GSTP1 couldinhibit MAPK activation in malignant cell lines indirectly through itsinteraction with TRAF2 or directly through its interaction with JNK1 andP38. These results together with the findings of the present inventionthat GSTP1 and TRAF2 are upregulated in patients with heart failure showthat GSTP1-TRAF2 complex formation is associated with the pathogenesisof heart failure. Tissue culture experiments in heart failure patientsrevealed that although the addition of TNF-α could suppress theanti-inflammatory effect of GSTP1, this effect can be retained byincreasing the GSTP1 dose. The anti-inflammatory properties of GSTP1were approved by the current results in vivo. The experimental modelused herein revealed that GSTP1 was able to ameliorate the inflammatoryreaction and decrease the cardiac remodeling after the induction of MI.Interestingly, GSTP1 was not only able to suppress the pro-inflammatorymediators like; IL-1, IL-2, JNK1, P38 and NF-kB but also could increasethe expression of the anti-inflammatory cytokine IL-10. Moreover, theresults according to the present invention revealed that GSTP1 protectsthe cardiac tissue against apoptosis in the failing heart. In concertwith this, it has been shown in oncological settings that GSTP1attenuates the autophosphorylation of TRAF2-enhanced apoptosissignal-regulating kinase1 (ASK1) and thus inhibits TRAF2-ASK1-inducedcell apoptosis by suppressing the interaction of TRAF2 and ASK1.

In conclusion, the present invention provides a novel function of GSTP1in modulating TNF-α/TRAF2 elicited JNK1/p38 activation in heart failure.The selective inhibition of TNF-α/TRAF2-mediated inflammatory signalingby GSTP1 was due to the present animal model shown to be a beneficialtreatment for patients with cardiomyopathies or ischemic heart diseases,especially for patients with myocardial infarction and heart failure.

The invention claimed is:
 1. A method for treating a human individual atrisk of developing cardiomyopathy (CMP) or ischemic heart disease (IHD)comprising: administering to said human individual glutathioneS-transferase P1 (GSTP1) in an amount effective to reduce the risk ofCMP and/or IHD, wherein the human individual is: a patient that has beendiagnosed with myocarditis or hypertension, a patient that has suffereda myocardial infarction, a patient that has been diagnosed with anginapectoris, a patient with heart insufficiency or heart failure, and/or apatient that suffers from a failing myocardium.
 2. The method of claim1, wherein the human individual is a patient that has been diagnosedwith myocarditis, or a patient that has been diagnosed withhypertension.
 3. The method of claim 2, wherein the myocarditis is acutemyocarditis; isolated myocarditis, myocarditis in a bacterial disease;myocarditis in a viral disease; acute or chronic myocarditis in Chagas'disease, myocarditis in toxoplasmosis, rheumatoid myocarditis or sarcoidmyocarditis.
 4. The method of claim 2, wherein the human individual is apatient that has been diagnosed with hypertension.
 5. The method ofclaim 4, wherein the patient is a stage 2 patient with systolic pressureof 160 mm Hg or more.
 6. The method of claim 4, wherein the patient hasdiastolic pressure of 100 mm Hg or more.
 7. The method of claim 2,wherein myocardial infarction has occurred in the patient.
 8. The methodof claim 1, wherein the human individual is a patient that has beendiagnosed with angina pectoris.
 9. The method of claim 8, whereinmyocardial infarction has occurred in the patient.
 10. The method ofclaim 8, wherein myocardial infarction has not occurred in the patient.11. The method of claim 1, wherein the human individual is a patientwith heart insufficiency or heart failure.
 12. The method of claim 11,wherein said GSTP1 is administered in an intravenous administration orin a continuous administration to the heart muscle.
 13. The methodaccording to claim 1, wherein the GSTP1 is parenterally administered.14. The method according to claim 1, wherein the GSTP1 is administeredin a dosage of 0.001 to 100 mg GSTP1/kg to the human individual.
 15. Themethod according to claim 1, wherein the GSTP1 is administered in adosage of 0.1 to 10000 U GSTP1/kg to the human individual.
 16. Themethod of claim 1, wherein the cardiomyopathy is dilated cardiomyopathy;obstructive hypertrophic cardiomyopathy; other hypertrophiccardiomyopathy; ischemic cardiomyopathy, endomyocardial (eosinophilic)disease; endocardial fibroelastosis; other restrictive cardiomyopathy;alcoholic cardiomyopathy, cardiomyopathy due to drugs and other externalagents, unspecified cardiomyopathies; cardiomyopathy in infectious andparasitic diseases; cardiomyopathy in metabolic diseases; cardiomyopathyin nutritional diseases; Gouty tophi of heart or thyrotoxic heartdisease.
 17. The method according to claim 16, wherein the acutemyocarditis is infective myocarditis; the myocarditis in a bacterialdisease is diphtheritic, gonococcal, meningococcal, syphilitic ortuberculous myocarditis; and the myocarditis in a viral disease isinfluenzal myocarditis or mumps myocarditis.
 18. The method according toclaim 17, wherein the infective myocarditis is septic myocarditis. 19.The method according to claim 13, wherein the GSTP1 is administeredintraperitoneally or intravenously.
 20. The method according to claim14, wherein the GSTP1 is administered in a dosage of 0.01 to 10 mgGSTP1/kg.
 21. The method according to claim 20, wherein the GSTP1 isadministered in a dosage of 0.1 to 1 mg GSTP1/kg via intravenousadministration.
 22. The method according to claim 15, wherein the GSTP1is administered in a dosage of 1 to 1000 U GSTP1/kg.
 23. The methodaccording to claim 22, wherein the GSTP1 is administered in a dosage of10 to 100 U GSTP1/kg via intravenous administration.
 24. The method ofclaim 1, wherein the human individual is a patient suffering from afailing myocardium.